Yeast Protocols Handbook User Manual FOR RESEARCH USE ONLY PT3024-1 (PR973283)

Transcription

User Manual
Yeast Protocols Handbook
FOR RESEARCH USE ONLY
United States/Canada
800.662.2566
Asia Pacific
+1.650.919.7300
Europe
+33.(0)1.3904.6880
Japan
+81.(0)77.543.6116
Clontech Laboratories, Inc.
A Takara Bio Company
1290 Terra Bella Ave.
Mountain View, CA 94043
Technical Support (US)
E-mail: tech@clontech.com
www.clontech.com
PT3024-1 (PR973283)
Published July 2009
Table of Contents
I. Introduction
4
II. Introduction to Yeast Promoters
5
III. Culturing and Handling Yeast
10
IV.
Preparation of Yeast Protein Extracts
A. General Information
B. Preparation of Yeast Cultures for Protein Extraction
C. Preparation of Protein Extracts: Urea/SDS Method
D. Preparation of Protein Extracts: TCA Method
E. Troubleshooting 12
12
12
13
15
17
V.
Yeast Transformation Procedures
B. Reagents and Materials Required
C. Tips for a Successful Transformation
D. Integrating Plasmids into the Yeast Genome
E. Small-scale LiAc Yeast Transformation Procedure
F. Troubleshooting Yeast Transformation 18
18
19
20
20
20
22
VI.
α- and β-Galactosidase Assays
B. In vivo Plate Assay Using X-gal in the Medium
C. Colony-lift Filter Assay D. Liquid Culture Assay Using ONPG as Substrate
E. Liquid Culture Assay Using CPRG as Substrate
F. Liquid Culture Assay Using a Chemiluminescent Substrate
G. α-Gal Quantitative Assay
23
23
26
26
27
28
29
32
VII.
Working with Yeast Plasmids A. General Information
B. Plasmid Isolation From Yeast
C. Transforming E. coli with Yeast Plasmids
34
34
34
36
VIII. Analysis of Yeast Plasmid Inserts by PCR A. General Information
B. Tips for Successful PCR of Yeast Plasmid Templates
39
39
39
IX.
42
42
43
44
Additional Useful Protocols
A. Yeast Colony Hybridization B. Generating Yeast Plasmid Segregants
C. Yeast Mating
X. References
46
PPENDICES
A
A. Glossary of Technical Terms B. Yeast Genetic Markers Used in the Matchmaker Systems
C. Media Recipes A. Yeast Media
B. E. coli Media
D. Solution Formulations
E. Plasmid Information
F. Yeast Host Strain Information
49
51
52
52
55
56
60
63
www.clontech.com
2 Protocol No. PT3024-1
Version No. PR973283
Table of Contents continued
List of Tables
Table I.
Yeast Promoter Constructs Used to Regulate Reporter Gene Expression
in Matchmaker Plasmids and Host Strains
6
Table II.
Yeast Promoter Constructs in the Matchmaker Cloning Vectors
9
Table III.
Comparison of β-galactosidase Assays
25
Table IV.
Selected Yeast Genes and Their Associated Phenotypes
51
Table V.
Matchmaker Reporter Genes and Their Phenotypes
51
Table VI.
Matchmaker Two-Hybrid System Cloning Vectors
60
Table VII.
Matchmaker Two-Hybrid System Reporter and Control Plasmids
61
Table VIII. Matchmaker One-Hybrid System Cloning, Reporter & Control Plasmids
62
Table IX.
63
Yeast Reporter Strains in the Matchmaker One- and Two-Hybrid Systems
List of Figures
Figure 1.
Figure 2.
Sequence of GAL4 DNA-BD recognition sites in the GAL1, GAL2, MEL1
UASs and the UASG 17-mer Urea/SDS protein extraction method
6
14
Figure 3.
TCA protein extraction method
16
Contact Us For Assistance
Customer Service/Ordering:
Technical Support:
Telephone: 800.662.2566 (toll-free)
Telephone: 800.662.2566 (toll-free)
Fax: 800.424.1350 (toll-free)
Fax: 650.424.1064
Web: www.clontech.com
Web: www.clontech.com
E-mail: orders@clontech.com
E-mail: tech@clontech.com
Notice to Purchaser
Clontech products are to be used for research purposes only. They may not be used for any other purpose, including,
but not limited to, use in drugs, in vitro diagnostic purposes, therapeutics, or in humans. Clontech products may not be
transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service
to third parties without written approval of Clontech Laboratories, Inc.
NOTICE TO PURCHASER: LIMITED LICENSE
Use of this product is covered
6,127,155. The purchase of this product includes a limited, non-transferable immunity from suit under
the foregoing patent claims for using only this amount of product for the purchaser’s own internal research. No right
under any other patent claim (such as method claims in U.S. Patents Nos.
5,994,056 and 6,171,785)
and no right to perform commercial services of any kind, including without limitation reporting the results of purchaser’s
activities for a fee or other commercial consideration, is hereby conveyed by the purchase of this product expressly,
by implication, or by estoppel. This product is for research use only. Diagnostic uses require a separate license from
Roche. Further information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied
Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA.
Clontech, the Clontech logo and all other trademarks are the property of Clontech Laboratories, Inc., unless noted
otherwise. Clontech is a Takara Bio Company. ©2009 Clontech Laboratories, Inc.
Protocol No. PT3024-1
www.clontech.com
3
I. Introduction
TheYeast Protocols Handbook provides background information and general yeast protocols that
complement our system-specific User Manuals.The protocols in this Handbook have been optimized
with our yeast-based Matchmaker™ Two-Hybrid and One-Hybrid Systems, and Matchmaker
Libraries. The Yeast Protocols Handbook is especially useful for researchers who wish to use
yeast as a vehicle for their molecular biology experiments, but have little or no prior experience
working with yeast. For novice and experienced users alike, the Yeast Protocols Handbook will
help you obtain the best possible results with your Matchmaker and other yeast-related products
from Clontech.
This Handbook includes:
• detailed information on culturing and handling yeast
• information on the yeast promoters used in the Matchmaker Systems
• two protocols for preparing protein extracts from yeast
• quantitative and qualitative β-galactosidase assays (for use with lacZ yeast reporter
strains)
• a simple, optimized protocol for isolating plasmids from yeast
• PCR amplification and yeast colony hybridization protocols for the rapid analysis of positive
clones obtained in a library screening
• a small-scale, lithium acetate yeast transformation protocol
• additional protocols for working with certain yeast plasmids and host strains
The special application of yeast transformation for one- and two-hybrid library screening is covered
in detail in each product-specific User Manual. The special application of yeast mating for library
screening is covered in the Pretransformed Matchmaker Libraries User Manual.
About our yeast-based products
The Matchmaker GAL4 Two-Hybrid Systems (Cat No. K1604-1, K1605-1, 630303) and LexA TwoHybrid System (Cat No. K1609-1) are complete kits for identifying and investigating protein-protein
interactions in vivo using the yeast two-hybrid assay. The Matchmaker One-Hybrid System (Cat
No. K1603-1) provides the basic tools for identifying novel proteins in vivo that bind to a target
DNA sequence such as a cis-acting regulatory element. Matchmaker Two-Hybrid Systems are
compatible with our pBridge Three-Hybrid Vector (Cat No. 630404) for the investigation of tertiary
protein complexes. The Matchmaker Libraries are constructed in vectors that express inserts as
fusions to a transcriptional activation domain, and are thus a convenient resource for researchers
wishing to screen a library using the one- or two-hybrid assays. Pretransformed Matchmaker
Libraries provide an even greater level of convenience for those wishing to perform a two-hybrid
library screening without using large- or library-scale yeast transformations.
Clontech offers an extensive line of kits and reagents that support and complement the
Matchmaker Systems and Libraries. The YeastmakerTM Yeast Transformation Kit (Cat No. 630439)
includes all the necessary reagents and protocols for efficient transformation using the lithium
acetate method. Also available from Clontech: a selection of GAL4 DNA-binding domain (DNA-BD)
and activation domain (AD) hybrid cloning vectors; the pGilda Vector for use with LexA-based
two-hybrid systems; monoclonal antibodies and sequencing primers; and yeast media, including
Minimal SD Base and many different formulations of Dropout (DO) Supplement. Finally, the pHACMV and pMyc-CMV Vector Set (Cat No. 631604) can be used to confirm protein interactions in
mammalian cells.
For ordering information on these products, please see Chapter XI of this Handbook or the
Clontech Catalog.
www.clontech.com
II. Introduction to Yeast Promoters
Yeast promoters and other cis-acting regulatory elements play a crucial role in yeast-based
expression systems and transcriptional assays such as the Matchmaker One- and Two-Hybrid
Systems. Differences in the promoter region of reporter gene constructs can significantly affect
their ability to respond to the DNA-binding domain of specific transcriptional activators; promoter
constructs also affect the level of background (or leakiness) of gene expression and the level of
induced expression. Furthermore, differences in cloning vector promoters determine the level of
protein expression and, in some cases, confer the ability to be regulated by a nutrient (such as
galactose in the case of the GAL1 promoter).
This chapter provides a brief introduction to several commonly used yeast promoters and cisregulatory elements. For further information on the regulation of gene expression in yeast, we
recommend the Guide to Yeast Genetics and Molecular Biology by Guthrie & Fink (1991; No.
V2010-1); Molecular Biology and Genetic Engineering of Yeasts, edited by Heslot & Gaillardin
(1992); Stargell & Struhl (1996); and Pringle et al. (1997; No. V2365-1).
UAS and TATA regions are basic building blocks of yeast promoters
The initiation of gene transcription in yeast, as in other organisms, is achieved by several
molecular mechanisms working in concert. All yeast structural genes (i.e., those transcribed by
RNA polymerase II) are preceded by a region containing a loosely conserved sequence (TATA
box) that determines the transcription start site and is also a primary determinant of the basal
transcription level. Many genes are also associated with cis-acting elements—DNA sequences
to which transcription factors and other trans-acting regulatory proteins bind and affect
transcription levels. The term “promoter” usually refers to both the TATA box and the associated
cis-regulatory elements.This usage is especially common when speaking of yeast gene regulation
because the cis regulatory elements are relatively closely associated with the TATA box (Yoccum,
1987). This is in contrast to multicellular eukaryotes, where cis-regulatory elements (such as
enhancers) can be found very far upstream or downstream from the promoters they regulate.
In this text, “minimal promoter” will refer specifically to the TATA region, exclusive of other
cis-acting elements.
The minimal promoter (or TATA box) in yeast is typically approximately 25 bp upstream of the
transcription start site. Yeast TATA boxes are functionally similar to prokaryotic Pribnow boxes,
but are not as tightly conserved. Furthermore, some yeast transcription units are preceded by
more than one TATA box. The yeast HIS3 gene, for example, is preceded by two different TATA
boxes: TR, which is regulated, and TC, which is constitutive (Mahadevan & Struhl, 1990). Yeast
TATA boxes can be moved to a new location, adjacent to other cis-regulatory elements, and still
retain their transcriptional function.
One type of cis-acting transcription element in yeast is upstream activating sequences (UAS),
which are recognized by specific transcriptional activators and enhance transcription from adjacent
downstream TATA regions. The enhancing function of yeast UASs is generally independent of
orientation; however, it is sensitive to distance effects if moved more than a few hundred base pairs
from the TATA region. There may be multiple copies of a UAS upstream of a yeast coding region.
In addition, UASs can be eliminated or switched to change the regulation of target genes.
UAS and TATA regions can be switched to create novel promoters
The “mix and match” nature of yeast TATA boxes and UASs has been used to great advantage in
yeast two-hybrid systems to create novel promoters for the reporter genes. (For general references
on yeast two-hybrid systems, see Chapter X.) In most cases, the lacZ, HIS3, ADE2 and LEU2
reporter genes are under control of artificial promoter constructs comprised of a TATA and UAS
(or operator) sequence derived from another gene (Table I). In some cases, the TATA sequence
and the UAS are derived from different genes; indeed, the LexA operator is a cis-acting regulatory
element derived from E. coli.
For GAL4-based systems, either a native GAL UAS or a synthetic UASG 17-mer consensus sequence
www.clontech.com
5
II. Introduction to Yeast Promoters continued
table i. yeast promoter constructs used to regulate reporter gene expression
in
Plasmid or
host straina
Reporter
gene
Matchmaker plasmids and host strains
Origin of
UAS UAS regulated by
Origin of
Expression levelb
TATA sequence Induced (uninduced)
CG-1945
lacZ
HIS3
UASG 17-mer (x3)c
GAL1
GAL4 GAL4 CYC1
GAL1
low
high(slightlyleaky)
HF7c
lacZ
HIS3
UASG 17-mer (x3)c
GAL1
GAL4
GAL4 CYC1
GAL1
low
high (tight)
Y190
lacZ
HIS3
GAL1
GAL1
GAL4
GAL4 GAL1
HIS3 (TC+TR)
high
high (leaky)
Y187
lacZ d
GAL1
GAL4 GAL1
high
SFY526
lacZ
GAL1
GAL4
GAL1
high
PJ69-2A
HIS3
ADE2
GAL1
GAL2
GAL4
GAL4
GAL1
GAL2
high (tight)
high (tight)
AH109
HIS3
ADE2
lacZ
GAL1
GAL2
MEL1
GAL4
GAL4
GAL4
GAL1
GAL2
MEL1
high (tight)
high (tight)
low
EGY48
LEU2
LexA op(x6)
LexA
LEU2
high
p8op-lacZ
lacZ
LexA op(x8)
LexA
GAL1 high
pHISi
HIS3
(none)f
(n.a.)
HIS3 (TC+TR)
n.a.f (leaky)
pHISi-1
HIS3
(none)f
(n.a.)
HIS3 (TC+TR)
n.a.f (leaky)
pLacZi
lacZ
(none)f
(n.a.)
CYC1
n.a.f (tight)
e
See Appendices E & F for references.
When induced by a positive two-hybrid interaction; “leaky” and “tight” refer to expression levels in the absence of
induction.
c
Conserved 17-bp palindromic sequence to which the GAL4 protein binds (Guthrie & Fink, 1991).
d
Y187 probably contains two copies of the lacZ gene, judging by the strength of the signal in this strain and in the strains
from which it was derived (Durfee et al., 1993; Harper et al., 1993).
e
This is the minimal TATA region of the GAL1 promoter; it does not include the GAL1 UAS and therefore is not responsive
to regulation by GAL4 protein.
f
The Matchmaker One-Hybrid System vectors do not contain a UAS because they are used to experimentally test target
elements inserted upstream of the minimal promoter for their ability to bind specific transcriptional activators. In the
absence of inserted target elements, reporter gene expression is not induced; however, expression levels may be leaky,
depending on the nature of the minimal promoter used in that vector.
a
b
GAL1 UAS
GAL1-bs1
GAL1-bs2
GAL1-bs3
GAL1-bs4
TAGAAGCCGCCGAGCGG
GACAGCCCTCCGAAGGA
GACTCTCCTCCGTGCGT
CGCACTGCTCCGAACAA
GAL2 UAS
GAL2-bs1
GAL2-bs2
GAL2-bs3
GAL2-bs4
GAL2-bs5
CGGAAAGCTTCCTTCCG
CGGCGGTCTTTCGTCCG
CGGAGATATCTGCGCCG
CGGGGCGGATCACTCCG
CGGATCACTCCGAACCG
MEL1 UAS
CGGCCATATGTCTTCCG
UAS G17-mer
CGGAAGACTCTCCTCCG
Figure 1. Sequence of the GAL4 DNA-BD recognition sites in the GAL1, GAL2, and MEL1 UASs and the UASG 17-mer
consensus sequence (Giniger & Ptashne, 1988).
www.clontech.com
(Heslot & Gaillardin, 1992) provides the binding site for the GAL4 DNA-BD. For LexA-based systems,
multiple copies of the LexA operator provide the binding site for the LexA protein. If you are
putting together your own one- or two-hybrid system, you must make sure that the reporter gene’s
promoter will be recognized by the DNA-BD moiety encoded in your DNA-BD fusion vector.
Reporter genes under the control of GAL4-responsive elements
In yeast, the genes required for galactose metabolism are controlled by two regulatory proteins,
GAL4 and GAL80, as well as by the carbon source in the medium (Guthrie & Fink, 1991; Heslot &
Gaillardin, 1992). When galactose is present, the GAL4 protein binds to GAL4-responsive elements
within the UAS upstream of several genes involved in galactose metabolism and activates
transcription. In the absence of galactose, GAL80 binds to GAL4 and blocks transcriptional activation.
Furthermore, in the presence of glucose, transcription of the galactose genes is immediately
repressed (Johnston et al., 1994).
The UASs of the 20 known galactose-responsive genes all contain one or more conserved
palindromic sequences to which the GAL4 protein binds (Guthrie & Fink, 1991; Giniger et al. 1985;
reviewed in Heslot & Gaillardin, 1992). The 17-mer consensus sequence, referred to here as UASG
17-mer, functions in an additive fashion, i.e., multiple sites lead to higher transcription levels than a
single site (Giniger & Ptashne, 1988). The protein binding sites of the GAL1, GAL2, MEL1 UASs,
and the UASG 17-mer consensus sequence, are shown in Figure 1.
The tight regulation of the GAL UASs by GAL4 makes it a valuable tool for manipulating expression
of reporter genes in two-hybrid systems that are dependent on the GAL4 DNA-BD. However, in
such systems, the yeast host strains must carry deletions of the gal4 and gal80 genes to avoid
interference by endogenous GAL4 and GAL80 proteins; thus, no significant glucose repression is
observed in these strains and no induction is observed unless a two-hybrid interaction is occurring.
Therefore, nutritional regulation of GAL UASs is not a feature of GAL4-based two-hybrid systems.
However, the host strain used in the LexA system does support galactose induction, as it is wild
type for GAL4 and GAL80 functions.
In the GAL4-based MatchmakerTwo-Hybrid Systems, either an intact GAL1, GAL2 or MEL1 UAS or
an artifically constructed UAS consisting of three copies of the 17-mer consensus binding sequence,
is used to confer regulated expression on the reporter genes (Table I). The HIS3 reporter of AH109,
PJ69-2A, HF7c, and CG-1945, and the lacZ reporter ofY190,Y187, and SFY526 are all tightly regulated
by the intact GAL1 promoter (including the GAL1 UAS and GAL1 minimal promoter). In HF7c and
CG1945, lacZ expression is under control of UASG 17-mer (x3) and the extremely weak minimal promoter
of the yeast cytochrome C1 (CYC1) gene. lacZ under the control of the intact GAL1 promoter can be
expressed at ~10X the level obtained with the UASG 17-mer (x3)/CYC1 minimal promoter construct under
similar induction conditons (Clontech Laboratories; unpublished data). Therefore, some weak or
transient two-hybrid interactions may not be detectable in HF7c or CG1945 unless you use a highly
sensitive β-galactosidase assay (such a liquid culture assay using a chemiluminescent substrate;
Chapter VI.F). The ADE2 reporter of PJ69-2A and AH109 is tightly regulated by the intact GAL2
promoter, whose induction properties are similar to those of the GAL1 promoter. In AH109, lacZ is
under the control of the MEL1 UAS and minimal promoter.The MEL1 promoter is stonger than the
UASG 17-mer (x3)/CYC1 minimal promoter, but weaker than the GAL1 promoter (Aho et al., 1997).
Reporter genes under the control of a minimal HIS3 promoter
The native yeast HIS3 promoter contains a UAS site recognized by the transcriptional activator
GCN4, and two TATA boxes. GCN4 regulates one of the TATA boxes (TR), while the other TATA box
(TC) drives low-level constitutive expression of HIS3 (Iyer & Struhl, 1995). TC is not regulated by
the native GCN4-binding UAS, the GAL1 UAS, or artificial UASG constructs (Mahadevan & Struhl,
1990; Hope & Struhl, 1986).
The HIS3 reporter gene in yeast strain Y190 is unusual among the GAL4 two-hybrid reporter gene
www.clontech.com
7
constructs in that it is under the control of the GAL1 UAS and a minimal promoter containing both
HIS3 TATA boxes (Flick & Johnston, 1990). The result is high-level expression (due to the GAL1
UAS) when induced by a positive two-hybrid interaction; this construct also exhibits a significant
level of constitutive leaky expression (due to the HIS3 TC). In contrast, in HF7c, CG-1945, PJ69-2A,
and AH109 the entire HIS3 promoter (including bothTATA boxes) was replaced by the entire GAL1
promoter, leading to tight regulation of the HIS3 reporter in those strains (Feilotter et al., 1994).
The HIS3 reporter plasmids pHISi and pHISi-1 used in the Matchmaker One Hybrid System also
have both of the HIS3 TATA boxes present in the minimal promoter. By inserting a cis-acting
element in the MCS, the regulated TATA box (TR) can be affected, but there is still a significant
amount of constitutive, leaky expression due to the HIS3 TC. The leaky HIS3 expression of these
one-hybrid plasmids is first used to help construct HIS3 reporter strains, and later is controlled
by including 3-aminotriazole in the medium to suppress background growth.
Reporter genes under the control of LexA operators
In LexA-based two-hybrid systems, the DNA-BD is provided by the entire prokaryotic LexA
protein, which normally functions as a repressor of SOS genes in E. coli when it binds to LexA
operators, which are an integral part of the promoter (Ebina et al., 1983). When used in the yeast
two-hybrid system, the LexA protein does not act as a repressor because the LexA operators are
integrated upstream of the minimal promoter and coding region of the reporter genes. LEU2
reporter expression in yeast strain EGY48 is under the control of six copies of the LexA operator
(op) sequence and the minimal LEU2 promoter. In the lacZ reporter plasmids, lacZ expression is
under control of 1–8 copies of the LexA op (Estojak et al., 1995) and the minimal GAL1 promoter.
Because all of the GAL1 UAS sequences have been removed from the lacZ reporter plasmids
(West et al., 1984), this promoter is not regulated by glucose or galactose.
Promoters used to drive fusion protein expression in two-hybrid cloning vectors
The ADH1 promoter (or a truncated version of it) is the promoter used to drive expression of the
fusion proteins in most of the Matchmaker cloning vectors.The 1500-bp full-length ADH1 promoter
(Ammerer, 1983; Vainio, GenBank accession number: Z25479) leads to high-level expression of
sequences under its control in pGADT7, pGAD GH, pLexA, and pAS2-1 during logarithmic growth
of the yeast host cells.Transcription is repressed in late log phase by the ethanol that accumulates
in the medium as a by-product of yeast metabolism.
Several Matchmaker cloning vectors contain a truncated 410-bp ADH1 promoter (Table II). At one
point, it was believed that only this portion was necessary for high-level expression (Beier &Young,
1982). In most vector constructs, however, this truncated promoter leads to low or very low levels of
fusion protein expression (Ruohonen et al., 1991; Ruohonen et al., 1995;Tornow & Santangelo, 1990).
This observation has been confirmed at Clontech by quantitative Western blots (unpublished data).
The high-level expression reported by Beier &Young (1982) was apparently due to a segment of DNA
derived from pBR322, which was later found to coincidentally enhance transcriptional activity in yeast
(Tornow & Santangelo, 1990). In the Matchmaker vector pACT2, strong constitutive fusion protein
expression is driven by the 410-bp truncated ADH1 promoter adjacent to this enhancing pBR322
segment.
The DNA-BD cloning vector pGBKT7 used in MatchmakerTwo-Hybrid System 3 contains a 700-bp
fragment of the ADH1 promoter. This trucated promoter leads to high-level expression, but no
ethanol repression (Ruohonen et al., 1991; Ruohonen et al., 1995).
The AD cloning vector pB42AD and the alternative DNA-BD vector pGilda used in the Matchmaker
LexATwo-Hybrid System utilize the full-length GAL1 promoter to drive fusion protein expression.
Because the LexA system host strain is wild-type for GAL4 and GAL80, fusion protein expression
is regulated by glucose and galactose.
www.clontech.com
table ii. yeast promoter constructs in the
Vectorsa
Promoter
Matchmaker cloning vectors
Regulation/
Relative Protein Expression Level
Signal
Strength on
Western blot b
p LexA, pGAD GH, pAS2-1, pAS2, pGADT7 ADH1 (full-length)
Ethanol-repressed/High
+++
pACT2, pACT
ADH1 (410 bp+)c
Constitutive/medium
++
pGAD GL
ADH1 (410 bp)
Constitutive/low
+/– (weak)
pGAD424, pGAD10
pGBT9
Constitutive/ very low
pGBKT7
Consitutive/high
ADH1 (700 bp)
(not detectable)
+++
pB42AD, pGilda
GAL1 (full-length)
Repressed by glucose;
(not detectable)d
induced (high-level) by galactose +++d
p8op-lacZ
GAL1 (minimal)
Not regulated by glucose
or galactose
(no data)
See Appendix E for vector references.
Unpublished data obtained at Clontech Laboratories using the appropriate GAL4 domain-specific mAb (Cat No. 630402 or
Cat No. 630403). Soluble protein extracts were prepared from CG-1945 transformed with the indicated plasmid. Samples
equivalent to ~1 OD600 unit of cells were electrophoresed and then blotted to nitrocellulose filters. The blots were probed
with either GAL4 DNA-BD mAb (0.5 µg/ml) or GAL4 AD mAb (0.4 µg/ml) using 1 ml of diluted mAb per 10 cm2 of blot,
followed by HRP-conjugated polyclonal Goat Anti-Mouse IgG (Jackson Immunological Research; diluted 1:15,000 inTBST).
Signals were detected using a chemiluminescent detection assay and a 2.5-min exposure of x-ray film. Signal intensities
were compared to that of known amounts of purified GAL4 DNA-BD (a.a. 1–147) or GAL4 AD (a.a. 768–881).
c
The truncated ADH1 promoter in pACT2 is adjacent to a section of pBR322 which acts as a transcriptional enhancer in
yeast.
d
Data obtained using EGY48[p8op-lacZ] transformed with pGilda and grown in the presence of glucose or galactose,
respectively (April 1997 Clontechniques); no data available for pB42AD.
a
b
www.clontech.com
9
III.Culturing and Handling Yeast
For additional information on yeast, we recommend Guthrie and Fink (1991) Guide toYeast Genetics
and Molecular Biology (Cat No. V2010-1).
A. Yeast Strain Maintenance, Recovery from Frozen Stocks, and Routine Culturing
1.Long-term storage
• Yeast strains can be stored indefinitely in YPD medium with 25% glycerol at –70°C. For
storage >1 year, the temperature must be maintained below –55°C.
• Transformed yeast strains are best stored in the appropriate SD dropout medium to
keep selective pressure on the plasmid. (See Appendix C.A for recipes and Appendix
E for plasmid information.)
To prepare new glycerol stock cultures of yeast:
a. Use a sterile inoculation loop to scrape an isolated colony from the agar plate.
b. Resuspend the cells in 200–500 µl of YPD medium (or the appropriate SD medium)
in a 1.5-ml microcentrifuge tube. Vortex tube vigorously to thoroughly disperse the
cells. Add sterile 50% glycerol to a final concentration of 25%.
c. Tightly close the cap. Shake the vial before freezing at –70°C.
2.To recover frozen strains and prepare working stock plates:
a. Streak a small portion of the frozen glycerol stock onto a YPD (or appropriate SD)
agar plate.
b. Incubate the plate at 30°C until yeast colonies reach ~2 mm in diameter (this takes
3–5 days). Use these colonies as your working stock.
c. Seal plates with Parafilm and store at 4°C for up to two months. Streak a fresh working
stock plate from the frozen stock at 1–2-month intervals.
d. If you cannot recover the strain, the cells may have settled ; in this case, thaw the
culture on ice, vortex vigorously, and restreak.The glycerol stock tube may be refrozen
a few times without damaging the cells.
3.To prepare liquid overnight cultures:
a. Use only fresh (<2-months old) colonies from the working stock plate. Use one large
(2–3-mm diameter) colony per 5 ml of medium. If colonies are small, or if you are
inoculating a larger volume, use several colonies. Important: Vigorously vortex the
medium for ~1 min to thoroughly disperse the cells.
Notes:
• Liquid cultures will grow slower than expected if clumps are present in the inoculum; cells in the
interior of the clumps do not have access to the nutrients in the medium.
• If you are inoculating a volume greater than 1 ml, it is easier to disperse the clumps if the colonies are
first placed in 1 ml of medium in a microcentrifuge tube, vortexed, and then transferred to the desired
volume.
• When growing overnight cultures of yeast transformants, use the appropriate SD minimal medium
to keep selective pressure on extrachromosomal plasmid(s).
• The growth inYPD of yeast strains carrying the ade2-101 mutation will be enhanced by adding adenine
hemisulfate (0.003% final concentration) to the medium (Appendix C.A). All of the host strains (except
EGY48) used in the Matchmaker Systems carry this auxotrophic mutation.
• The growth of transformed PJ69-2A cells in SD/–Trp may also be enhanced by adding excess adenine
to the medium (Appendix C.A).
b. Incubate at 30°C for 16–18 hr with shaking at 230–270 rpm. With most strains, this
will yield a stationary phase culture (OD600 > 1.5).
Note: Different yeast strains grow at different rates. Growth rates may also be affected by the presence
of fusion proteins in certain transformants. In addition, the doubling time of most strains growing in SD
minimal medium is twice as long as in YPD.
c. If you need a mid-log phase culture, transfer enough of the overnight culture into
fresh medium to produce an OD600 = 0.2–0.3. Incubate at 30°C for 3–5 hr with shaking
(230–250 rpm). This will, with most strains, produce a culture with an OD 600
~0.4–0.6.
Note: Generally, YPD or YPDA may be used in this incubation. Because of the shorter incubation time,
plasmid loss will not be significant. However, do not use YPD if you want to induce protein expression
from the yeast GAL1 promoter of a LexA system plasmid, e.g., pB42AD or pGilda; YPD contains glucose,
which represses transcription from the GAL1 promoter.
www.clontech.com
III. Culturing and Handling Yeast continued
B. Growth Selection for Transformation Markers and Reporter Gene Expression
Most yeast cloning vectors and control plasmids (including those provided in our Matchmaker
Systems) carry at least one nutritional marker to allow for selection of yeast transformants
plated on SD minimal medium lacking that specific nutrient. Furthermore, if you are
cotransforming yeast with two or more different plasmids bearing different nutritional
markers, the plasmids can be independently selected. Thus, the SD selection medium you
choose for plating transformants depends generally on the purpose of the selection. Specific
factors to consider in choosing the appropriate SD selection medium are:
• the plasmid(s) used and whether you are selecting for one or more plasmids
• whether you are selecting for colonies in which two hybrid proteins are interacting
• whether—and to what extent—the host strain is leaky for reporter gene expression
• whether you want to induce protein expression from the regulated GAL1 promoter
• whether you intend to perform in-vivo, agar-plate β-galactosidase assays (for lacZ
reporter expression in the LexA Two-Hybrid System).
Please refer to your system-specific User Manual for further information on choosing the
appropriate SD selection media for particular plasmids, host strains, and applications.
www.clontech.com
11
IV.Preparation of Yeast Protein Extracts
We provide two alternative protocols for the preparation of protein extracts from yeast. The
results (i.e., protein yield and quality) will vary depending on the protein and may be more
successful with one protocol than with the other. Because it is difficult to predict which
procedure will give better results, we provide two protocols for comparison. The cell culture
preparation method (Section B) is the same for both protein extraction procedures.
Both extraction procedures address the two most challenging aspects of isolating proteins
from yeast: 1) disrupting yeast cell walls; and 2) inhibiting the many endogenous yeast
proteases. Yeast cell walls are tough and must be disrupted by a combination of physical
and chemical means; methods that utilize glycolytic enzymes are not recommended for this
application because they are often contaminated with proteases. Endogenous proteases
must be counteracted with a cocktail of strong protease inhibitors (recipe in Appendix
D.A). If you know your protein of interest is susceptible to a protease not inhibited by the
recommended cocktail, add the appropriate inhibitor before using the mixture.You may also
wish to add other inhibitors such as sodium fluoride to prevent dephosphorylation, if that
is appropriate for your protein.
B. Preparation of Yeast Cultures for Protein Extraction
Reagents and Materials Required:
• YPD and appropriate SD liquid medium (Recipes in Appendix C.A)
• 20- and 50-ml culture tubes
• Ice-cold H2O
• Dry ice or liquid nitrogen
1.For each transformed yeast strain you wish to assay in a Western blot, prepare a 5-ml
overnight culture in SD selection medium as described in Section III.A, except use a
single isolated colony (1–2 mm in diameter, no older than 4 days). Use the SD medium
appropriate for your system and plasmids (Appendix E). Also prepare a 10-ml culture
of an untransformed yeast colony in YPD or (if possible) appropriate SD medium as a
negative control.
2.Vortex the overnight cultures for 0.5–1 min to disperse cell clumps. For each clone to be
assayed (and the negative control), separately inoculate 50-ml aliquots of YPD medium
with the entire overnight culture.
3.Incubate at 30°C with shaking (220–250 rpm) until the OD600 reaches 0.4–0.6. (Depending
on the fusion protein, this will take 4–8 hr.) Multiply the OD600 (of a 1-ml sample) by the
culture volume (i.e., 55 ml) to obtain the total number of OD600 units; this number will
be used in Sections C & D. (For example, 0.6 x 55 ml = 33 total OD600 units.)
Note: During late log phase the ADH1 promoter shuts down and the level of endogenous yeast proteases
increases.
4.Quickly chill the culture by pouring it into a prechilled 100-ml centrifuge tube halfway
filled with ice.
5.Immediately place tube in a prechilled rotor and centrifuge at 1000 x g for 5 min at
4°C.
6.Pour off supernatant and resuspend the cell pellet in 50 ml of ice-cold H2O. (Any unmelted
ice pours off with the supernatant.)
7.Recover the pellet by centrifugation at 1,000 x g for 5 min at 4°C.
8.Immediately freeze the cell pellet by placing the tube on dry ice or in liquid nitrogen.
Store cells at –70°C until you are ready to proceed with the experiment.
www.clontech.com
IV. Preparation of Yeast Protein Extracts continued
C. Preparation of Protein Extracts: Urea/SDS Method
(Figure 2; Printen & Sprague, 1994)
• 1.5-ml screw-cap microcentrifuge tubes
• Glass beads (425–600 µm; Sigma Cat No. G-8772)
• Protease inhibitor solution (Appendix D.A)
• PMSF stock solution (Appendix D.A)
• Cracking buffer stock solution (Appendix D.A)
• Cracking buffer, complete (Appendix D.A)
Note: Unless otherwise stated, keep protein samples on ice.
1.Prepare complete cracking buffer (Appendix D.A) and prewarm it to 60°C. Because
the PMSF degrades quickly, prepare only the amount of cracking buffer you will need
immediately. Use 100 µl of cracking buffer per 7.5 OD600 units of cells. (For example, for
33 total OD600 units of cells, use 0.44 ml of cracking buffer.)
2.Quickly thaw cell pellets by separately resuspending each one in the prewarmed cracking
buffer.
• If cell pellets are not immediately thawed by the prewarmed cracking buffer, place the
tubes briefly at 60°C to hasten melting. To avoid risk of proteolysis, do not leave them
longer than 2 min at 60°C.
• Because the initial excess PMSF in the cracking buffer degrades quickly, add an
additional aliquot of the 100X PMSF stock solution to the samples after 15 min and
approximately every 7 min thereafter until Step 9, when they are placed on dry ice
or are safely stored at –70°C or colder. (Use 1 µl of 100X PMSF per 100 µl of cracking
buffer.)
3.Transfer each cell suspension to a 1.5-ml screw-cap microcentrifuge tube containing 80
µl of glass beads per 7.5 OD600 units of cells.
Note: The volume of the glass beads can be measured using a graduated 1.5-ml microcentrifuge tube.
4.Heat samples at 70°C for 10 min.
Note: This initial incubation at 70°C frees membrane-associated proteins.Thus, if you skip this step, membraneassociated proteins will be removed from the sample at Step 6 (high-speed centrifugation).
5.Vortex vigorously for 1 min.
6.Pellet debris and unbroken cells in a microcentrifuge at 14,000 rpm for 5 min, preferably
at 4°C, otherwise at room temperature (20–22°C).
7.Transfer the supernatants to fresh 1.5-ml screw-cap tubes and place on ice (first
supernatants).
8.Treat the pellets as follows:
a. Place tubes in a 100°C (boiling) water bath for 3–5 min.
b. Vortex vigorously for 1 min. c. Pellet debris and unbroken cells in a microcentrifuge at 14,000 rpm for 5 min,
preferably at 4°C, otherwise at room temperature.
d. Combine each supernatant (second supernatant) with the corresponding first
supernatant (from Step 7).
Note: If no supernatant is obtained, add more cracking buffer (50–100 µl) and repeat Steps 8.b & c.
9.Boil the samples briefly. Immediately load them on a gel. Alternatively, samples may be
stored on dry ice or in a –70°C freezer until you are ready to run them on a gel.
www.clontech.com
13
Cell pellets
• Thaw and resuspend cell pellets
in prewarmed Cracking buffer
• Add cells to glass beads
• Heat at 70°C for 10 min
• Vortex vigorously for 1 min
• Centrifuge at 14,000 rpm for 5 min
Pellet
First supernatant
• Place on ice
• Boil for 3–5 min
• Vortex vigorously for 1 min
• Centrifuge at 14,000 rpm for
for 5 min
Pellet
(discard)
Second
supernatant
• Combine with
second
supernatant
• Place on ice
Combined supernatants
• Immediately load gel or
freeze at –70°C or colder
Figure 2. Urea/SDS protein extraction method.
www.clontech.com
D. Preparation of Protein Extracts: TCA Method
(Figure 3; Horecka, J., personal communication)
• 1.5-ml screw-cap microcentrifuge tubes
• Glass beads (425–600 µm; Sigma Cat No. G-8772)
• Protease inhibitor solution (Appendix D.A)
• PMSF Stock solution (Appendix D.A; Add as necessary throughout the protocol.)
• [Recommended] Bead Beater (BioSpec, Bartlesville, OK)
Note: If you do not have access to a Bead Beater, a high-speed vortexer can be used instead. However, vortexing
is not as effective as bead-beating at disrupting the cells.
• TCA buffer (Appendix D.A)
• Ice-cold 20% w/vTCA in H2O (see Sambrook et al. [1989] for tips on preparingTCA solutions)
• TCA-Laemmli loading buffer (Appendix D.A)
Note: Unless otherwise stated, keep protein samples on ice.
1.Thaw cell pellets on ice (10–20 min).
2.Resuspend each cell pellet in 100 µl of ice-cold TCA buffer per 7.5 OD600 units of cells. (For
example, for 33 total OD600 units of cells, use 0.44 ml of TCA buffer.) Place tubes on ice.
3.Transfer each cell suspension to a 1.5-ml screw-cap microcentrifuge tube containing
glass beads and ice-cold 20% TCA. Use 100 µl of glass beads and 100 µl of ice-cold 20%
TCA per 7.5 OD600 units of cells.
Note: The volume of the glass beads can be measured using a graduated 1.5-ml microcentrifuge tube.
4.To disrupt cells, place tubes in a Bead-Beater and set speed at highest setting. Bead-beat
the cells for 2 X 30 sec, placing tubes on ice for 30 sec in between the two bead-beatings.
Place tubes on ice.
Note: If you do not have access to a Bead-Beater, you can vortex the tubes vigorously at 4°C for 10 min; alternatively,
you can vortex at room temperature for shorter periods (of 1 min each) at least 4 times, placing tubes on ice for
30 sec in between each vortexing. Place tubes on ice.
5.Transfer the supernatant above the settled glass beads to fresh 1.5-ml screw-cap tubes
and place tubes on ice. This is the first cell extract.
Note: The glass beads settle quickly, so there is no need to centrifuge tubes at this point.
6.Wash the glass beads as follows:
a. Add 500 µl of an ice-cold, 1:1 mixture of 20% TCA and TCA buffer.
b. Place tubes in Beat Beater and beat for another 30 sec at the highest setting.
(Alternatively, vortex for 5 min at 4°C, or vortex 2 X 1 min at room temperature,
placing the tube on ice for 30 sec in between the two vortexings.)
c. Transfer the liquid above the glass beads (second cell extract) to the corresponding
first cell extract from Step 5.
7.Allow any carryover glass beads to settle in the combined cell extracts ~1 min, then
transfer the liquid above the glass beads to a fresh, prechilled 1.5-ml screw-cap tube.
8.Pellet the proteins in a microcentrifuge at 14,000 rpm for 10 min at 4°C.
9.Carefully remove supernatant and discard.
10.Quickly spin tubes to bring down remaining liquid. Remove and discard liquid using a
pipette tip.
11.Resuspend each pellet in TCA-Laemmli loading buffer. Use 10 µl of loading buffer per
OD600 unit of cells.
Note: If too much acid remains in the sample, the bromophenol blue in the buffer will turn yellow. Generally,
this will not affect the results of the electrophoresis.
12.Place tubes in a 100°C (boiling) water bath for 10 min.
13.Centrifuge samples at 14,000 rpm for 10 min at room temperature (20–22°C).
14.Transfer supernatant to fresh 1.5-ml screw-cap tube.
15.Load the samples immediately on a gel. Alternatively, samples may be stored on dry
ice or in a –70°C freezer until you are ready to run them on a gel.
www.clontech.com
15
Cell pellets
• Thaw and resuspend cell pellets
in cold TCA buffer
• Add cells to glass beads and ice-cold, 20% TCA
• Bead-beat cells 2 x 30 sec
(or vortex vigorously for 10 min at 4°C)
First Cell Extract
(liquid above beads)
Beads
and unbroken cells
• Add ice-cold 20% TCA
• Bead-beat cells 1 x 30 sec
(or vortex for 5 min at 4°C)
Beads
and unbroken cells
(discard)
Second Cell Extract
• Place on ice
• Combine
Cell Extracts
• Allow glass beads
to settle ~1 min
Beads
Combined Cell Extracts
and unbroken cells
(discard)
Pellet
(Protein and contaminants)
Supernatant (Discard)
• Resuspend in TCA-Laemmli loading buffer
• Boil 10 min
Pellet (discard)
Supernatant
(Protein extract)
Immediately load gel or
freeze at –70°C or colder
Figure 3. TCA protein extraction method.
www.clontech.com
E. Troubleshooting
Optimal electrophoretic separation of proteins depends largely on the quality of the equipment
and reagents used in the gel system, the manner in which the protein samples are prepared
prior to electrophoresis, the amount of protein loaded on the gel, and the voltage conditions
used during electrophoresis. These same considerations are important for the subsequent
transfer of proteins to the nitrocellulose membrane where transfer buffer composition,
temperature, duration of transfer, and the assembly of the blotting apparatus can all have
profound effects on the quality of the resultant protein blot. The following troubleshooting
tips pertain to the isolation of protein from yeast. Information on running polyacrylamide
protein gels and performing Western blots is available in published laboratory manuals (e.g.,
Sambrook et al., 1989, or Ausubel et al., 1987–96).
1.Few or no immunostained protein bands on the blot
• The transfer of protein bands to the blot may be confirmed by staining the blot with
Ponceau S.
• The presence of protein bands in the gel (before transfer) may be confirmed by
staining a parallel lane of the gel with Coomassie blue. (Note that once a gel has
been stained with Coomassie blue, the protein bands will not transfer to a blot.)
• The extent of cell wall disruption can be determined by examining a sample of treated
cells under the microscope. Incomplete cell lysis will lower the protein yield.
2.Several bands appear on the blot where a single protein species is expected
• Protein degradation and/or proteolysis may have occurred during sample preparation.
Additional protease inhibitors may be used as desired. Also, make sure that in Steps
C.8.a and D.12 (boiling the protein extracts), the samples are placed into a water bath
that is already boiling. If samples are placed in the water before it has reached boiling
temperature, a major yeast protease (Proteinase B) will be activated. (Proteinase B
is a serine protease of the subtilisin family.)
• Dephosphorylation of a normally phosphoryated fusion protein may have occurred
during sample preparation. Sodium fluoride (NaF) may be added to the protease
inhibitor stock solution to help prevent dephosphorylation (Sadowski et al., 1991).
3.If you are running a reducing gel, make sure that the protein sample has been completely
reduced with either dithiothreitol or 2-mercaptoethanol prior to loading the gel.
www.clontech.com
17
V. Yeast Transformation Procedures
LiAc-mediated yeast transformation
There are several methods commonly used to introduce DNA into yeast, including the
spheroplast method, electroporation, and the lithium acetate (LiAc)-mediated method
(reviewed in Guthrie & Fink, 1991). At Clontech, we have found the LiAc method (Ito et al.,
1983), as modified by Schiestl & Gietz (1989), Hill et al. (1991), and Gietz et al. (1992), to be
simple and highly reproducible. This chapter provides detailed protocols for using the LiAc
procedure in a standard plasmid transformation and in a modified transformation to integrate
linear DNA into the yeast genome.
In the LiAc transformation method, yeast competent cells are prepared and suspended in
a LiAc solution with the plasmid DNA to be transformed, along with excess carrier DNA.
Polyethylene glycol (PEG) with the appropriate amount of LiAc is then added and the mixture
of DNA and yeast is incubated at 30°C. After the incubations, DMSO is added and the cells
are heat shocked, which allows the DNA to enter the cells. The cells are then plated on the
appropriate medium to select for transformants containing the introduced plasmid(s).
Because, in yeast, this selection is usually nutritional, an appropriate synthetic dropout (SD)
medium is used.
Simultaneous vs. sequential transformations
The LiAc method for preparing yeast competent cells typically results in transformation
efficiencies of 105 per µg of DNA when using a single type of plasmid. When the yeast is
simultaneously cotransformed with two plasmids having different selection markers, the
efficiency is usually an order of magnitude lower due to the lower probability that a particular
yeast cell will take up both plasmids. (Yeast, unlike bacteria, can support the propagation
of more than one plasmid having the same replication origin, i.e., there is no plasmid
incompatibility issue in yeast.) Thus, in a cotransformation experiment, the efficiency of
transforming each type of plasmid should remain at ~105 per µg of DNA, as determined by
the number of colonies growing on SD medium that selects for only one of the plasmids.
The cotransformation efficiency is determined by the number of colonies growing on SD
medium that selects for both plasmids and should be ~104 cfu/µg DNA.
Simultaneous cotransformation is generally preferred because it is simpler than sequential
transformation—and because of the risk that expression of proteins encoded by the first plasmid
may be toxic to the cells. If the expressed protein is toxic, clones arising from spontaneous
deletions in the first plasmid will have a growth advantage and will accumulate at the expense
of clones containing intact plasmids. However, if there is no selective disadvantage to cells
expressing the first cloned protein, sequential transformation may be preferred because it
uses significantly less plasmid DNA than simultaneous cotransformation. In some cases,
such as when one of the two plasmids is the same for several different cotransformations,
sequential transformations may be more convenient.
Scaling up or down
The small-scale yeast transformation procedure described here can be used for up to 15 parallel
transformations, and uses 0.1 µg of each type of plasmid. Depending on the application, the
basic yeast transformation method can be scaled up without a decrease in transformation
efficiency. If you plan to perform a two-hybrid library screening, you will need a large or
library-scale transformation procedure, which will require significantly more plasmid DNA.
Please refer to your Matchmaker system-specific User Manual for further information on
library screening strategies and specific protocols.
Integration vs. nonintegration of yeast plasmids
For most yeast transformations performed while using the Matchmaker Systems, it is not
necessary or desirable to have the plasmid integrate into the yeast genome. (In fact, yeast
plasmids do not efficiently integrate if they carry a yeast origin of replication and are used
uncut.) However, there are two exceptions to this general rule, as explained in the respective
system-specific User Manuals: (a) In the Matchmaker One-Hybrid System, the researcher must
www.clontech.com
V. Yeast Transformation Procedures continued
construct their own custom reporter plasmid and then integrate it into the yeast host strain
before performing the one-hybrid assay. (b) In the Matchmaker LexA Two-Hybrid System,
the p8op-lacZ reporter plasmid can be used either as an autonomously replicating plasmid
or as an integrated plasmid, depending on the desired level of reporter gene expression.The
primary reason for integrating a plasmid in some Matchmaker applications is to generate a
stable yeast reporter strain in which only one copy of the reporter gene is present per cell,
and thereby control the level of background expression. If you have an application that
requires integration of a plasmid into the yeast genome, please see Section V.D.
Transformation controls
When setting up any type of transformation experiment, be sure to include proper controls
for transformation efficiencies. In the case of simultaneous cotransformation, it is important
to determine the transformation efficiencies of both plasmids together, as well as of each
type of plasmid independently. That way, if the cotransformation efficiency is low, you may
be able to determine whether one of the plasmid types was responsible (seeTroubleshooting
Guide, Section F). Therefore, be sure to plate an aliquot of the transformation mixture on the
appropriate SD media that will select for only one type of plasmid. Example calculations are
shown in Section V.E. When screening a library or performing a one- or two-hybrid assay,
you will n
eed additional controls, as explained your system-specific User Manual.
B. Reagents and Materials Required
Note: The Yeastmaker Yeast Transformation System (Cat No. 630439) contains all the solutions (except media, H2O,
and DMSO) required for yeast transformation.Yeastmaker reagents have been optimized for use in the Matchmaker
One- and Two-Hybrid Systems.
• YPD or the appropriate SD liquid medium
• Sterile 1X TE/1X LiAc (Prepare immediately prior to use from 10X stocks; stock recipes in
Appendix D.B)
• Sterile 1.5-ml microcentrifuge tubes for the transformation
• Appropriate SD agar plates (100-mm diameter)
Notes:
• Prepare the selection media and pour the required number of agar plates in advance. (See your systemspecific User Manual or Appendix E for media recommendations.) Be sure to plan for enough plates for the
control transformations and platings.
• Allow SD agar plates to dry (unsleeved) at room temperature for 2–3 days or at 30°C for 3 hr prior to plating
any transformation mixtures. Excess moisture on the agar surface can lead to inaccurate results due to
uneven spreading of cells or localized variations in additive concentrations.
• Appropriate plasmid DNA in solution (check amounts required)
• Appropriate yeast reporter strain for making competent cells (check volume of competent
cells required; Steps 1–11 of Section V.E will give you 1.5 ml, enough for 14–15 small-scale
transformations)
• Carrier DNA (Appendix D.B)
• Sterile PEG/LiAc solution (Prepare only the volume needed, immediately prior to use, from
10X stocks; Appendix D.B)
• 100% DMSO (Dimethyl sulfoxide; Sigma Cat No. D-8779)
• Sterile 1X TE buffer (Prepare from 10X TE buffer; Appendix D.B)
• Sterile glass rod, bent Pasteur pipette, or 5-mm glass beads for spreading cells on plates.
www.clontech.com
19
C. Tips for a Successful Transformation
• Fresh (one- to three-week-old) colonies will give best results for liquid culture inoculation.
A single colony may be used for the inoculum if it is 2–3 mm in diameter. Scrape the entire
colony into the medium. If colonies on the stock plate are smaller than 2 mm, scrape
several colonies into the medium. See Chapter III.A for further information on starting
liquid cultures from colonies and from a liquid culture inoculum.
• Vigorously vortex liquid cultures to disperse the clumps before using them in the next
step.
• The health and growth phase of the cells at the time they are harvested for making
competent cells is critical for the success of the transformation. The expansion culture
(Step E.6) should be in log-phase growth (i.e., OD600 between 0.4 and 0.6) at the time the
cells are harvested. If they are not, see the Troubleshooting guide (Section V.F).
• When collecting cells by centrifugation, a swinging bucket rotor results in better recovery
of the cell pellet.
• For the highest transformation efficiency (as is necessary for library screening), use
competent cells within 1 hr of their preparation. If necessary, competent cells can be
stored (after Step E.11) at room temperature for several hours with a minor reduction in
competency.
• To obtain an even growth of colonies on the plates, continue to spread the transformation
mixtures over the agar surface until all liquid has been absorbed. Alternatively, use 5-mm
sterile glass beads (5–7 beads per 100-mm plate) to promote even spreading of the
cells.
D. Integrating Plasmids into the Yeast Genome
Important: Please read Section V.A for guidelines on when it is appropriate to use this
procedure.
To promote integration of yeast plasmids, follow the small-scale LiAc transformation procedure
(Section V.E below) with the following exceptions:
• Before transformation, linearize 1–4 µg of the reporter vector by digesting it with an
appropriate restriction enzyme in a total volume of 40 µl at 37°C for 2 hr. Electrophorese
a 2-µl sample of the digest on a 1% agarose gel to confirm that the plasmid has been
efficiently linearized.
Notes:
• If the vector contains a yeast origin of replication (i.e., 2 µ ori), it will be necessary to remove it before you
attempt to integrate the vector.
• The vector should be linearized within the gene encoding the transformation (i.e., nutritional selection) marker.
However, if the digestion site is within a region that is deleted in the host strain, the plasmid will not be able
to integrate. Please refer to your product-specific User Manual for recommended linearization sites.
• At Step 12, add 1–4 µg of the linearized reporter plasmid + 100 µg of carrier DNA; for each
reporter plasmid, also set up a control transformation with undigested plasmid (+ 100 µg
carrier DNA).
• At Step 20, resuspend cells in 150 µl of TE buffer.
• Plate the entire transformation mixture on one plate of the appropriate SD medium to
select for colonies with an integrated reporter gene.
E. Small-scale LiAc Yeast Transformation Procedure
1.Inoculate 1 ml of YPD or SD with several colonies, 2–3 mm in diameter.
Note: For host strains previously transformed with another autonomously replicating plasmid, use the
appropriate SD selection medium to maintain the plasmid (Appendix E).
2.Vortex vigorously for 5 min to disperse any clumps.
3.Transfer this into a flask containing 50 ml of YPD or the appropriate SD medium.
4.Incubate at 30°C for 16–18 hr with shaking at 250 rpm to stationary phase (OD600>1.5).
5.Transfer 30 ml of overnight culture to a flask containing 300 ml ofYPD. Check the OD600 of
the diluted culture and, if necessary, add more of the overnight culture to bring the OD600
up to 0.2–0.3.
www.clontech.com
6.Incubate at 30°C for 3 hr with shaking (230 rpm). At this point, the OD600 should be
0.4–0.6.
Note: If the OD600 is <0.4, something is wrong with the culture (see Troubleshooting Section F.6).
7.Place cells in 50-ml tubes and centrifuge at 1,000 x g for 5 min at room temperature
(20–21°C).
8.Discard the supernatants and thoroughly resuspend the cell pellets in sterileTE or distilled
H2O. Pool the cells into one tube (final volume 25–50 ml).
9.Centrifuge at 1,000 x g for 5 min at room temperature.
10.Decant the supernatant.
11.Resuspend the cell pellet in 1.5 ml of freshly prepared, sterile 1X TE/1X LiAc.
12.Add 0.1 µg of plasmid DNA and 0.1 mg of carrier DNA to a fresh 1.5-ml tube and mix.
Notes:
• For simultaneous cotransformation (using two different plasmids), use 0.1 µg of each plasmid (an
approximately equal molar ratio), in addition to the 0.1 mg of carrier DNA.
• For transformations to integrate a reporter vector, use at least 1 µg of linearized plasmid DNA in addition
to the carrier DNA.
13.Add 0.1 ml of yeast competent cells to each tube and mix well by vortexing.
14.Add 0.6 ml of sterile PEG/LiAc solution to each tube and vortex at high speed for 10 sec to
mix.
15.Incubate at 30°C for 30 min with shaking at 200 rpm.
16. Add 70 µl of DMSO. Mix well by gentle inversion. Do not vortex.
17.Heat shock for 15 min in a 42°C water bath.
18.Chill cells on ice for 1–2 min.
19.Centrifuge cells for 5 sec at 14,000 rpm at room temperature. Remove the
supernatant.
20.Resuspend cells in 0.5 ml of sterile 1X TE buffer.
21.Plate 100 µl on each SD agar plate that will select for the desired transformants.To ensure that
you will obtain a plate with well-separated colonies, also spread 100 µl of a 1:1000, 1:100, and
1:10 dilution on 100-mm SD agar plates.These will also provide controls for (co)transformation
efficiency.
Note: If you are performing a cotransformation, plate controls to check transformation efficiency and markers
of each plasmid. On separate 100-mm plates, spread 1 µl (diluted in 100 µl H2O) on medium that will select for
a single type of plasmid.
22.Incubate plates, up-side-down, at 30°C until colonies appear (generally, 2–4 days).
23.To calculate the cotransformation efficiency, count the colonies (cfu) growing on the
dilution plate from Step 22 above that has 30–300 cfu.
cfu x total suspension vol. (µl)
= cfu/µg DNA
Vol. plated (µl) x dilution factor x amt. DNA used (µg)*
* In a cotransformation, this is the amount of one of the plasmid types, not the sum of them. If you have used
unequal amounts of two plasmids, use the amount of the lesser of the two.
Sample calculation:
• 100 colonies grew on the 1:100 dilution plate (dilution factor = 0.01)
• plating volume: 100 µl
• resuspension volume = 0.5 ml
• amount of limiting plasmid = 0.1 µg
100 cfu x 0.5 ml x 103 µl/ml
= 5 x 105 cfu/µg DNA
100 µl x 0.01 x 0.1 µg
24. Pick the largest colonies and restreak them on the same selection medium for master
plates. Seal plates with Parafilm and store at 4°C for 3–4 weeks.
www.clontech.com
21
F. Troubleshooting Yeast Transformation
The overall transformation efficiency should be at least 104 cfu/µg for transformation
with a single type of plasmid, and 103 cfu/µg for simultaneous cotransformation with two
types of plasmids. If your cotransformation efficiency is lower than expected, calculate the
transformation efficiency of the single plasmids from the number of transformants growing
on the appropriate control plates. If the two types of plasmids separately gave transformation
efficiencies >105 cfu/µg, switch to sequential transformation.
If the transformation efficiency for one or both of the separate plasmids is <105 cfu/µg, several
causes are possible.
1.Suboptimal plasmid preparation
• Repeat the transformation using more (up to 0.5 µg) of the plasmid DNA that had the low
transformation efficiency.
• Check the purity of the DNA and, if necessary, repurify it by ethanol precipitation before
using it again.
2. Suboptimal carrier DNA
• If you are not already doing so, use Yeastmaker Carrier DNA, which is available separately
(Cat No. 630440) or as part of theYeastmakerYeastTransformation System (Cat No. 630439),
and has been optimized for high transformation efficiencies in this system.
• If transformation efficiencies are declining in successive experiments, the carrier DNA may
be renaturing. Reboil the carrier DNA for 20 min, and then chill it quickly in an ice-water
bath.
3. Suboptimal yeast competent cells
• Make sure that the expansion culture (Step E.6) was in log-phase growth at the time the
cells were harvested for making competent cells. If the overnight culture (Step E.4) or
expansion culture (Step E.6) grew slower than expected (or not at all), start over at Step
E.1 by preparing a fresh overnight culture. Failure to thoroughly disperse the colony used
for the inoculum will result in slow growth; see Section III.A.3. If you still have problems
obtaining a healthy liquid culture, streak a fresh working stock plate (from the frozen
glycerol stock) and inoculate with a fresh colony.
• Check the liquid medium to make sure it was made correctly. If you suspect that the
medium or carbon source stock solutions have been over-autoclaved, remake fresh
solutions and either filter sterilize them or adjust the autoclave settings appropriately
before autoclaving.
• The addition of adenine hemisulfate to YPD (in Steps E.3 and E.5) will enhance the growth
of yeast strains that contain the ade2-101 mutation. All of our Matchmaker host strains
(except EGY48) carry this mutation.
• Check the concentration of the resuspended competent cells (after Step E.11) using a
hemocytometer. If the cell concentration is <1 x 109/ml, spin the cells down again (at 1,000
x g for 5 min) and resuspend them in a smaller volume of 1X TE/LiAc buffer.
• Occasionally, there is a contaminant in the water that can affect transformation efficiency
and/or cell growth. Prepare all reagents using sterile, deionized, distilled water such as
Milli-QTM-filtered. Confirm that your water purification system is functioning properly.
www.clontech.com
VI. α- and β-Galactosidase Assays
Considerations
• To reduce variability in liquid assays, assay five separate transformant colonies, and
perform each assay in triplicate.
• It is important that the colonies to be assayed for α- and β-galactosidase activity
are growing on the appropriate SD minimal medium. SD (dropout) medium is
used to keep selective pressure on the hybrid plasmids and, in the case of the
Matchmaker LexA Two-Hybrid System, the lacZ reporter plasmid up to the time
the cells are lysed for the assay. The type of SD medium needed depends on the
plasmids and host strains used. Furthermore, when working with a lacZ reporter
under the control of the inducible GAL1 promoter (such as in the LexA System),
the SD medium must contain galactose (not glucose) as the carbon source. See the
system-specific User Manual for media recommendations.
β-Galactosidase Assays
• X-gal must be used as the β-galactosidase substrate for solid-support assays because
of its high degree of sensitivity. (X-gal is ~106-fold more sensitive than ONPG.) Although
more sensitive than X-gal, Galacton-StarTM is not recommended for agar plate and filter
assays because it gives troublesome background.
• The filter and liquid β-galactosidase assays described here use at least one freeze/thaw cycle
in liquid nitrogen to lyse the yeast cell walls. Freeze-thaw cycles are a rapid and effective cell
lysis method which permits accurate quantification of β-galactosidase activity (Schneider et
al., 1996).
• The colony-lift filter assay (Breeden & Nasmyth, 1985) used to measure β-galactosidase
activity is primarily used to screen large numbers of cotransformants that survive the
HIS3 growth selection in a GAL4 two-hybrid or one-hybrid library screeening. It can also
be used to assay for an interaction between two known proteins in a GAL4 two-hybrid
system.
• The in vivo, agar plate assay is primarily used to screen large numbers of cotransformants
for the expression of the lacZ reporter gene in a LexA two-hybrid library screening when
the reporter gene is maintained on an autonomously replicating plasmid.The in vivo assay
works for LexA transformants because of the lacZ reporter plasmid’s high copy number
and because of the preamplification step that normally precedes the β-galactosidase
assay in this system. (Please refer to the Matchmaker LexA Two-Hybrid User Manual
for more information on library screening.) Because of its relatively low sensitivity, the
in vivo, agar plate assay is not suitable for screening transformants in a GAL4-based
two-hybrid assay, or in a LexA-based two-hybrid assay when the reporter gene has been
integrated into the host genome.
• Liquid cultures are assayed for β-galactosidase to verify and quantify two-hybrid interactions.
Because of their quantitative nature, liquid assays can be used to compare the relative
strength of the protein-protein interactions observed in selected transformants. However,
there is no direct correlation between β-galactosidase activity and the Kd of an interaction
(Estojak et al., 1995). Furthermore, quantitative data cannot be compared between different
host strains having different lacZ reporter constructs. In fact, due to promoter strength
differences, it may be possible to quantitate the relative strength of interactions in some
yeast strains (e.g., Y190, Y187), but not in others (e.g., CG-1945 or HF7c). (See Chapter II
for a discussion of the promoters.)
• The liquid assays described here use one of three substrates: ONPG, CPRG, or a
chemiluminescent substrate (Galacton-Star). The three substrates differ in their relative
cost, sensitivity, and reproducibility. See Table III.
α-Galactosidase Assays
• The α-Gal Quantitative Assay is a sensitive colorimetric method for the detection and
quantitation of yeast α-galactosidase activity resulting from expression of the MEL1
reporter gene in our GAL4-based Matchmaker two-hybrid systems.
MEL1 is a member of the GAL gene family, which, as a group, facilitates the uptake
and utilization of galactose by the cell. Upon binding of GAL4 to the MEL1 upstream
activating sequence (MEL1 UAS), the MEL1 gene product, α-galactosidase, is actively
www.clontech.com
23
VI. α- and β-Galactosidase Assays continued
expressed and secreted to the periplasmic space and culture medium where it catalyzes
the hydrolysis of melibiose to galactose and glucose.The α-Gal Quantitative Assay allows
you to specifically identify and measure this catalytic activity using p-nitrophenyl-α-dgalactoside (PNP-α-Gal), a colorless compound that yields a yellow product (p-nitrophenol)
upon hydrolysis.
The α-Gal Quantitative Assay can be used to measure the extracellular α-galactosidase
activity produced during the culture of any yeast strain that carries the MEL1 gene. MEL1 is
endogenous to many but not all yeast strains. (Liljeström, 1985; Post-Beittenmiller et al., 1984).
Table IX provides a list of the GAL4-based Matchmaker yeast strains known to carry the MEL1
gene.This list includes strain AH109 used in Clontech’s MatchmakerTwo-Hybrid System 3
(Cat No. 630303) and included with Clontech’s GAL4-based cDNA libraries.
MEL1 has been shown to be a sensitive in vivo reporter for GAL4-based two-hybrid
assays (Aho et al., 1997). The quantitative nature of the α-Gal Assay makes it possible to
compare the degree of MEL1 expression in different Matchmaker two-hybrid host cell
populations containing different pairs of interacting proteins, or to measure differences
in the relative strength of binding between mutant forms of interacting proteins.
Principle of the α-Gal Quantitative Assay
In the α-Gal Quantitative Assay, the catalytic activity of α-galactosidase is monitored
colorimetrically by measuring the rate of hydrolysis of the chromogenic substrate,
p-nitrophenyl-α-d-galactoside (PNP-α-Gal). One of the products of this reaction,
p-nitrophenol, displays a strong absorption band at 410 nm.
PNP-α-Gal + H2O
α-galactosidase
p-nitrophenol +
D-galactose
(λmax = 410 nm)
Because yeast naturally secrete α-galactosidase into the surrounding medium, it is more
convenient to assay than β-galactosidase, an intracellular enzyme encoded by the lacZ
reporter gene. The α-galactosidase assay is carried out by simply combining a small
aliquot of cell-free culture media with a fixed volume of Assay Buffer; cell lysis is not
necessary. After a prescribed incubation time (usually 60 min), the absorbance at 410
nm (OD410), which is proportional to moles p-nitrophenol liberated, is recorded and used
to calculate the concentration of α-galactosidase in milliunits/(ml x cell).
• To monitor MEL1 expression directly on nutritional selection plates, use X-α-Gal (Cat
No. 630407).
X-α-Gal can be included in the medium before pouring plates or spread on top of
the medium before plating liquid cultures. As α-galactosidase accumulates in the
medium, it hydrolyzes
X-α-Gal causing yeast colonies to turn blue. Instructions for preparing X-α-Gal
indicator plates are given in the X-α-Gal Protocol-at-a-Glance (PT3353-2) supplied with
each purchase of the substrate. Directions for use can also be downloaded from our
website at www.clontech.com.
www.clontech.com
table iii. comparison of β-galactosidase assays
Type of assay Substrate
Protocol
Section Applications/Comments
In vivo,
X-gal in medium VI.B
• Less sensitive than colony-lift assays; agar plate recommended only when the cells to be assayed contain many copies of the lacZ reporter gene
(such as on a high-copy-number plasmid)
• Convenient for large-scale experiments; screen many plates and colonies at the same time
• Potential drawbacks:
– Qualitative results only
– Expensive if assaying many plates
– Need to check for blue color development at several time intervals between 24 and 96 hr.
– Background can be troublesome
Colony-lift,
X-gal on filter
VI.C
filter
• Relatively sensitive; recommended when the cells to be assayed contain one or only a few copies of the lacZ reporter gene
• Convenient for large-scale experiments; screen many plates and colonies at the same time
• Relatively inexpensive to screen many plates
• Get results quickly (in most cases, within a few hours)
– Qualitative results only
– More manipulations required than for in vivo assay
Liquid culture ONPG
VI.D
• For assaying a small number of selected transformants
• Less expensive than CPRG or Galacton-StarTM
– May not be sensitive enough to quantify weak or transient two-hybrid interactions
Liquid culture CPRG
VI.E
• For assaying a small number of selected transformants
• 10-times more sensitive than ONPG
– Less reproducible than ONPG for strong positive colonies because of CPRG’s fast reaction rate
Liquid culture ChemiluminescentVI.F
(Galacton-StarTM) • For assaying a small number of selected transformants
• The most sensitive β-gal substrate
– Relatively expensive
– Requires luminometer or scintillation counter
– Can give high background
Summary: Relative sensitivity of the five types of β-galactosidase assays:
[Least sensitive]
X-gal ONPG (in agar plates) (liquid assay)
[Most sensitive]
CPRG (liquid assay) X-gal (filter assay)
www.clontech.com
Galacton Star (liquid assay)
25
B. In vivo Plate Assay Using X-gal in the Medium (For LexA Systems only)
• Appropriate SD agar plates containing X-gal (80 mg/L) and 1X BU salts (Appendix C.A).
Notes:
• BU salts are included in the medium to maintain the optimum pH for β-galactosidase and to provide the
phosphate needed for the assay.
• The X-gal should be incorporated into the medium before the plates are poured. If the X-gal is spread over
the surface of the agar plates, it can result in uneven distribution and thus localized variations in X-gal
concentration. Also, the extra liquid on the plate surface (from spreading the X-gal) may lead to uneven
spreading of the cell suspension and will delay absorption of the liquid.
• X-gal is heat-labile and will be destroyed if added to hot (i.e.>55°C) medium.
• Prepare the required number of plates in advance. Allow plates to dry (unsleeved) at room temperature for
2–3 days or at 30°C for 3 hr prior to spreading or streaking the cells. Excess moisture on the agar surface
can lead to uneven spreading of cells.
1.Streak, replica plate, or spread the transformants to be assayed on selection medium
containing X-gal and BU salts.
• When performing a two-hybrid library screening where very few of the cotransformants
are expected to be positive for lacZ expression (or where it is difficult to predict the
number of interactors), plate the cells at a high density. We recommend plating at two
different densities to cover a range; e.g., 0.5 x 106 cfu on some (150-mm plates) and 2
x 106 on others.
• When performing a two-hybrid assay where most or all of the individual colonies may
be LacZ+, spread 200–400 cfu per 100-mm plate.
2.Incubate plates at 30°C for 4–6 days.
3.Check plates every 12 hr (up to 96 hr) for development of blue color.
Notes:
• If you are performing a two-hybrid library screening using the Matchmaker LexA System, please see the
User Manual for further information on identifying and storing LacZ+ colonies.
• Colonies grown on X-gal-containing medium will be somewhat smaller than those grown without X-gal.
C. Colony-lift Filter Assay
• Whatman No. 5 or VWR Grade 410 paper filters, sterile
Notes:
• 75-mm filters (e.g., VWR Cat No. 28321-055) can be used with 100-mm plates; 125-mm filters (e.g., VWR Cat No.
28321-113) can be used with 150-mm plates
• Alternatively, 85- and 135-mm filters can be specially ordered from Whatman.
• Nitrocellulose filters also can be used, but they are prone to crack when frozen.
•
•
•
•
•
Forceps for handling the filters
Z buffer (Appendix D)
Z buffer/X-gal solution (Appendix D)
X-gal stock solution (Appendix D)
Liquid nitrogen
1.For best results use fresh colonies (i.e., grown at 30°C for 2–4 days), 1–3 mm in
diameter.
Notes:
• If only a few colonies are to be assayed, streak them (or spread them in small patches) directly onto master
SD selection agar plates. Incubate the plates at 30°C for an additional 1–2 days, and then proceed with the
β-galactosidase assay below.
• Use the SD selection medium appropriate for your system and plasmids. When testing LexA transformants,
be sure to use gal/raff induction medium.
2.Prepare Z buffer/X-gal solution as described in Appendix D.
3.For each plate of transformants to be assayed, presoak a sterile Whatman No. 5 or VWR
grade 410 filter by placing it in 2.5–5 ml of Z buffer/X-gal solution in a clean 100- or
150-mm plate.
www.clontech.com
4.Using forceps, place a clean, dry filter over the surface of the plate of colonies to be
assayed. Gently rub the filter with the side of the forceps to help colonies cling to the
filter.
5.Poke holes through the filter into the agar in three or more asymmetric locations to orient
the filter to the agar.
6.When the filter has been evenly wetted, carefully lift it off the agar plate with forceps and
transfer it (colonies facing up) to a pool of liquid nitrogen. Using the forceps, completely
submerge the filters for 10 sec.
Note: Liquid nitrogen should be handled with care; always wear thick gloves and goggles.
7.After the filter has frozen completely (~10 sec), remove it from the liquid nitrogen and
allow it to thaw at room temperature. (This freeze/thaw treatment is to permeabilizes
the cells.)
8.Carefully place the filter, colony side up, on the presoaked filter (from Step C.3). Avoid
trapping air bubbles under or between the filters.
9.Incubate the filters at 30°C (or room temperature) and check periodically for the appearance
of blue colonies.
Notes: • The time it takes colonies producing β-galactosidase to turn blue varies, typically from 30 min to 8 hr in a
library screening. Prolonged incubation (>8 hr) may give false positives.
• Yeast transformed with the β-galactosidase positive control plasmid will turn blue within 20–30 min. Most
yeast reporter strains cotransformed with the positive controls for a two-hybrid interaction give a positive
blue signal within 60 min. CG-1945 cotransformed with the control plasmids may take an additional 30 min
to develop. If the controls do not behave as expected, check the reagents and repeat the assay.
10.Identify the β-galactosidase-producing colonies by aligning the filter to the agar plate
using the orienting marks. Pick the corresponding positive colonies from the original
plates to fresh medium. If the entire colony was lifted onto the filter, incubate the original
plate for 1–2 days to regrow the colony.
D.
Liquid Culture Assay Using ONPG as Substrate
• Appropriate liquid medium (Appendix C.A)
• 50-ml culture tubes
• Z buffer (Appendix D)
• Z buffer + β-mercaptoethanol (Appendix D)
• ONPG (Appendix D)
• 1 M Na2CO3
• Liquid nitrogen
1.Prepare 5-ml overnight cultures in liquid SD selection medium as described in Chapter
III.A.3. Use the SD medium appropriate for your system and plasmids.
Note: Be sure to use SD medium that will maintain selection on the plasmids used.
2.On the day of the experiment, dissolve ONPG at 4 mg/ml in Z buffer (Appendix D) with
shaking for 1–2 hr.
3.Vortex the overnight culture tube for 0.5–1 min to disperse cell clumps. Immediately
transfer 2 ml of the overnight culture to 8 ml of YPD (except for the LexA System).
Note: For the LexA System, use the appropriate SD/Gal/Raff induction medium for the strains being
assayed.
4.Incubate the fresh culture at 30°C for 3–5 hr with shaking (230–250 rpm) until the cells
are in mid-log phase (OD600 of 1 ml = 0.5–0.8). Record the exact OD600 when you harvest
the cells.
Note: Before checking the OD, vortex the culture tube for 0.5–1 min to disperse cell clumps.
5.Place 1.5 ml of culture into each of three 1.5-ml microcentrifuge tubes. Centrifuge at
14,000 rpm (10,000 x g) for 30 sec.
6.Carefully remove supernatants. Add 1.5 ml of Z buffer to each tube and vortex until cells
are resuspended.
7.Centrifuge cells again and remove supernatants. Resuspend each pellet in 300 µl of Z
www.clontech.com
27
buffer. (Thus, the concentration factor is 1.5 /0.3 = 5-fold).
Note: Differences in cell recoveries after this wash step can be corrected for by re-reading the OD600 of the
resuspended cells.
8.Transfer 0.1 ml of the cell suspension to a fresh microcentrifuge tube.
9.Place tubes in liquid nitrogen until the cells are frozen (0.5–1 min).
10.Place frozen tubes in a 37°C water bath for 0.5–1 min to thaw.
11.Repeat the freeze/thaw cycle (Steps 9 & 10) two more times to ensure that the cells have
broken open.
12.Set up a blank tube with 100 µl of Z buffer.
13.Add 0.7 ml of Z buffer + β-mercaptoethanol to the reaction and blank tubes. Do not add
Z buffer prior to freezing samples.
14.Start timer. Immediately add 160 µl of ONPG in Z buffer to the reaction and blank
tubes.
15.Place tubes in a 30°C incubator.
16.After the yellow color develops, add 0.4 ml of 1 M Na2CO3 to the reaction and blank tubes.
Record elapsed time in minutes.
Notes:
•
The time needed will vary (3–15 min for the single-plasmid, β-gal-positive control; ~30 min for a two-hybrid
positive control; and up to 24 hr for weaker interactions).
•
The yellow color is not stable and will become more intense with time. You will need to run a new blank
tube with every batch.
17.Centrifuge reaction tubes for 10 min at 14,000 rpm to pellet cell debris.
18.Carefully transfer supernatants to clean cuvettes.
Note: The cellular debris, if transferred with the supernatant, will strongly interfere with the accuracy of this
test.
19.Calibrate the spectrophotometer against the blank at A420 and measure the OD420 of the samples
relative to the blank. The ODs should be between 0.02–1.0 to be within the linear range of the
assay.
20.Calculate β-galactosidase units. 1 unit of β-galactosidase is defined as the amount which
hydrolyzes 1 µmol of ONPG to o-nitrophenol and D-galactose per min per cell (Miller,
1972; Miller, 1992):
β-galactosidase units= 1,000 x OD420 /(t x V x OD600)
where:
t = elapsed time (in min) of incubation
V = 0.1 ml x concentration factor*
OD600 = A600 of 1 ml of culture
* The concentration factor (from Step D.7) is 5. However, it may be necessary to try several dilutions of
cells at this step (hence different concentration factors) to remain within the linear range of the assay.
E. Liquid Culture Assay Using CPRG as Substrate
• Buffer 1 (Appendix D)
• Buffer 2 (Appendix D)
• CPRG (chlorophenol red-β-D-galactopyranoside; Roche Applied Science Cat. No.10884308001)
• 3 mM ZnCl2 (Filter sterilized to preserve for ~3 months)
• Liquid nitrogen
1.Prepare 5-ml overnight cultures in liquid SD medium as described in Chapter III.A.3. Use the
SD selection medium appropriate for your system and plasmids.
Note: Be sure to use SD medium that will maintain selection on the plasmids used.
2.Vortex the overnight culture tube for 0.5–1 min to disperse cell clumps. Immediately
www.clontech.com
transfer 2 ml of the overnight culture to 8 ml of YPD (except for LexA System).
assayed.
3.Incubate fresh culture at 30°C for 3–5 hr with shaking (230–250 rpm) until the cells are
in mid-log phase (OD600 of 1 ml = 0.5–0.8). Record the exact OD600 when you harvest the
cells.
Note: Before checking the OD, vortex the culture tube for 0.5–1 min to disperse cell clumps.
14,000 rpm (16,000 x g) for 30 sec to pellet the cells.
5.Carefully remove the supernatant, add 1.0 ml of Buffer 1, and vortex until cells are
thoroughly resuspended.
6.Centrifuge at 14,000 rpm (16,000 x g) for 30 sec to pellet the cells.
7. Carefully remove the supernatant and resuspend the cells in 300 µl of Buffer 1.
(The concentration factor is 1.5 /0.3 = 5-fold.)
Note: Differences in cell recoveries after this wash step can be corrected for by re-reading the OD600 of the
resuspended cells.
8.Transfer 0.1 ml of the cell suspension to a fresh microcentrifuge tube.
9.Place tubes in liquid nitrogen until the cells are frozen (0.5–1 min).
11.Repeat the freeze/thaw cycle (Steps 9 and 10) two times to ensure that all cells are broken
open.
12.Add 0.7 ml of Buffer 2 to each sample and mix by vortexing. Thorough mixing is critical
to the assay.
13.Record the time when Buffer 2 was added. This is the starting time.
14.Add 1 ml of Buffer 2 to a separate tube (this will be the buffer blank).
15.When the color of the samples is yellow/grey to red, add 0.5 ml of 3.0 mM ZnCl2 to each
sample and the buffer blank to stop color development. Record the stop time. (For very strong
β-galactosidase-positive colonies, color development occurs within seconds; weak-tomoderate reactions take several hours to develop).
16.Centrifuge samples at 14,000 rpm for 1 min to pellet cell debris.
17.Transfer samples to fresh tubes.
18.Zero the spectrophotometer using the buffer blank and measure the OD578 of the samples.
(An OD578 between 0.25 and 1.8 is within the linear range of the assay.)
19.Calculate β-galactosidase units. 1 unit of β-galactosidase is defined as the amount which
hydrolyzes 1 µmol of CPRG to chlorophenol red and D-galactose per min per cell (Miller,
1972; Miller, 1992):
β-galactosidase units= 1000 x OD578 /(t x V x OD600)
where:
V = 0.1 x concentration factor*
OD600 = A600 of 1 ml of culture
* The concentration factor (from Step E.7) is 5. However, it may be necessary to try several dilutions of
cells at this step (hence different concentration factors) to remain within the linear range of the assay.
F. Liquid Culture Assay Using a Chemiluminescent Substrate
• Z buffer (Appendix D)
• Galacton-Star reaction mixture (Provided with the Luminescent β-galactosidase Detection Kit II)
• Liquid nitrogen
• Luminometer [or scintillation counter with single-photon-counting program]
• Optional: 96-well, opaque white, flat-bottom microtiter plates [Xenopore Cat No. WBP005]
www.clontech.com
29
• Optional: Purified β-galactosidase (for a standard curve)
Note: For best results, we recommend using the Luminescent β-galactosidase Detection Kit II (Cat No. 631712), which
includes a reaction buffer containing the Galacton-Star substrate and the Sapphire IITM accelerator, positive control bacterial
β-galactosidase, and a complete User Manual.
Chemiluminescent detection of β-galactosidase
It is important to stay within the linear range of the assay. High-intensity light signals can saturate
the photomultiplier tube in luminometers, resulting in false low readings. In addition, low
intensity signals that are near background levels may be outside the linear range of the assay. If in
doubt, determine the linear range of the assay and, if necessary, adjust the amount of lysate used
to bring the signal within the linear range. See Campbell et al. (1995) for a chemiluminescent
β-galactosidase assay used in a yeast two-hybrid experiment.
1.Prepare 5-ml overnight cultures in liquid SD medium as described in Chapter III.A.3. Use
the SD medium appropriate for your system and plasmids.
Note: For qualitative data, a whole colony, resuspended in Z buffer, may be used for the assay directly. See
instructions following this section.
2.On the day of the experiment, prepare the Galacton-Star reaction mixture. Keep buffer
on ice until you are ready to use it.
3.Vortex the overnight culture tube for 0.5–1 min to disperse cell clumps. Immediately transfer
at least 2 ml of the overnight culture to no more than 8 ml of YPD (except for the LexA
System).
assayed.
4.Incubate the fresh culture at 30°C for 3–5 hr with shaking (230–250 rpm) until the cells
are in mid-log phase (OD600 of 1 ml = 0.4–0.6).
5.Vigorously vortex the culture tube for 0.5–1 min to disperse cell clumps. Record the exact
OD600 when you harvest the cells.
14,000 rpm (10,000 x g) for 30 sec.
7.Carefully remove supernatants. Add 1.5 ml of Z buffer to each tube and thoroughly
resuspend the pellet.
8.Centrifuge at 14,000 rpm (10,000 x g) for 30 sec.
9. Remove the supernatants. Resuspend each pellet in 300 µl of Z buffer. (Thus, the
concentration factor is 1.5 /0.3 = 5-fold.)
10.Read the OD600 of the resuspended cells. The OD600 should be ~2.5. If the cell density is
lower, repeat Steps 5–9 , except resuspend the cells in <300 µl of Z buffer.
11.Vortex each cell suspension and transfer 100 µl to a fresh tube.
Note: The remaining cell suspension can be stored at –70°C to –80°C.
12.Place tubes in liquid nitrogen for 0.5–1 min to freeze the cells.
14.Repeat freeze/thaw cycle (Steps 12 & 13) once to ensure that cells have been cracked
open.
15.Warm to room temperature enough reaction buffer for the entire experiment.
16.Set up a blank tube with 25 µl of Z buffer.
17.[Optional] If you wish to obtain absolute as well as relative data, set up a series of
β-galactosidase standard tubes containing 0.0005, 0.001, 0.003, 0.010, and 0.020 unit of
β-galactosidase in 25 µl of Z-buffer.
18.Place 20–30 µl of each cell lysate in a separate sample tube (or into wells of an opaque
96-well, flat-bottom microtiter plate suitable for plate luminometers). If you are using a
sample tube, the tube should hold at least 0.5 ml.
Note: The amount of yeast extract required may vary depending upon the level of β-gal expression and the
detection device used. Use 10–30 µl of extract for positive controls and 20–30 µl for experimental samples
with potentially low levels of enzyme activity. It is important to vary the amount of extract to keep the signal
within the linear range of the assay.
19.Add 200 µl of Galacton-Star reaction mixture to each sample tube or well and mix
www.clontech.com
gently.
20.Incubate at room temperature (20°–25°C) for 60 min.
Note: Light signals produced during this incubation are stable for >1 hr; therefore, detection can be performed
1–2 hr after the incubation.
21.Centrifuge tubes at 14,000 rpm (16,000 x g) for 1 min at 4°C. (If you are using microtiter
plates, centrifuge plates at 1,000 x g for 5 min in a specially adapted rotor.) Proceed
directly to the appropriate detection steps for your assay: Step 22, 23, 24, or 27.
22.Detection using a tube luminometer
a. Turn on the tube luminometer. Set the integration time for 5 sec.
b. Calibrate the luminometer according to the manufacturer’s instructions.
c. If the sample is not already in a tube suitable for luminometer readings, transfer the
entire solution from (Step 21) to an appropriate tube. Do not disturb the pellet.
d. Place one sample at a time in the luminometer compartment and record the light emission
(RLU) as a 5-sec integral. Use your blank sample as a reference when interpreting the
data.
23.Detection using a plate luminometer
After Step 21, simply record light signals as 5-sec integrals.
24.Detection using a scintillation counter
a. Transfer the entire solution from Step 21 to a 0.5-ml microcentrifuge tube.
Note: Plan to use scintillation counter adaptors that keep the tubes upright.
b. Place the tube in the washer of the scintillation counter adaptor and place the adaptor
in the machine’s counting rack. Set the integration time for at least 15 sec.
Note: Integration times <15 sec may not produce accurate results.
c. To detect chemiluminescent signals, use a single-photon-count program. Consult your
scintillation counter’s manufacturer for further information about this software.
25.For detection methods described in Steps 22–24: Calculate the β-galactosidase activity in
terms of RLU/OD600 unit of cell culture. (Note that Miller unit calculations are not possible
using these methods.)
26.[Optional] If you have set up β-galactosidase standards, prepare a standard curve of RLU
vs. the amount of β-galactosidase. Estimate the quantity of β-galactosidase in the unknown
samples using the standard curve. Determine the amount of enzyme per OD600 unit of
cell culture. The final OD600 units of cells assayed per sample is calculated as follows:
OD600 (from Step 5) x vol (from Step 18) x conc. factor (from Step 9)
27.Detection by exposure of x-ray film
Light emission can also be recorded by exposure of x-ray film to reaction samples in
opaque 96-well flat-bottom microtiter plates. The relative intensity of the resulting spots
on the film can be estimated by comparison to positive and negative controls. Note that
x-ray film is several orders of magnitude less sensitive than a luminometer or scintillation
counter.
Overlay the microtiter plate with x-ray film, cover the film with plastic wrap, and place a heavy
object such as a book on top to hold the film in place. Expose the film at room temperature
for 5–30 min.
Note: To compare samples accurately, they must be within the linear response capability of the x-ray film. We
therefore recommend that you obtain several different exposures.
Qualitative Liquid Assay Using Galacton-Star as the Substrate
This alternative cell preparation method directly detects β-galactosidase activity in resuspended
yeast colonies. It is recommended for detecting extremely weak lacZ transcriptional signals
that cannot be detected by X-gal filter assays. For a +/– result, it is more labor-intensive than a
filter assay. However, because of its greater sensitivity, it is less likely to give a false-negative
result.
1.Grow colonies on the appropriate SD selection medium.
2.Transfer an entire large (2–3 mm), fresh (2–4-day-old) colony to a 0.5-ml tube containing
50 µl of Z buffer. If colonies are small, use several. At the same time, prepare a master
www.clontech.com
31
G.
or reference plate of the colonies to be assayed.
3.Completely resuspend the colony in the Z buffer by repeatedly pipetting up and down.
4.Place tubes in liquid nitrogen for 0.5–1 min to freeze the cells.
5.Continue with Step 13 of the main procedure (VI.F) above.
6.Compare results with those of the negative control.
α-Gal Quantitative Assay
• Appropriate liquid synthetic dropout (SD) culture medium (Appendix C.A)
• PNP-α-Gal Solution (100 mM)
• 10X Stop Solution (Appendix D.F)
• 1X NaOAc Buffer (Appendix D.F)
• Assay Buffer (Appendix D.F)
• 1.5-ml cuvettes or 96-well, flat-bottom microtiter plates for OD410 measurements
Preparation of Samples
1.Inoculate 2–5 ml of liquid synthetic dropout (SD) medium, containing the appropriate
dropout supplements, with a yeast colony expressing the pair of proteins being analyzed.
It is advisable to set up triplicate cultures for each type of yeast colony being analyzed.
Fresh (one- to three-week-old) colonies will give best results for liquid culture inoculation.
A single colony may be used for the inoculation if it is 2–3 mm in diameter. Scrape the
entire colony into the medium. If the colonies on the master plate are smaller than 2
mm, transfer several colonies into the medium.
Examples:
• For AH109 and Y190 cotransformants expressing interacting pairs of GAL4 BD and AD fusion proteins,
inoculate into SD/–His/–Leu/–Trp.
• For AH109 andY190 cotransformants expressing non-interacting pairs of fusion protein constructs, inoculate
into SD/–Leu/–Trp.
• For Y187 tranformants expressing interacting or non-interacting proteins, inoculate into SD/–Leu/–Trp.
• For non-transformed AH109, Y187, and Y190 host strains, inoculate into SD/–Ura.
2.Incubate at 30°C overnight (~16–18 hr) with shaking (250 rpm).
3.Vortex the cell culture tube for 0.5–1 min to disperse cell clumps, then transfer 1 ml of
the suspension to a clean cuvette and record the OD600. For accuracy, the OD600 should
lie between 0.5–1.0. Dilute the cell suspension if necessary; remember to account for the
dilution factor when making your final calculations (Step 11, below).
4.Place 1.0 ml of culture into a 1.5-ml microcentrifuge tube. Centrifuge at 14,000 rpm
(10,000 x g) for 2 min or until the cells are completely pelleted.
Note: It is important to ensure that cells and cellular debris are completely pelleted to minimize intereference
from light scattering in the colorimetric assay below.
5.Carefully transfer the supernatant to a clean tube and store at room temperature for use in
Step 6, below.
Note: To minimize the loss of enzyme activity, we suggest proceeding with the colorimetric assay immediately
once the cell-free superantant has been isolated.
Colorimetric Assay
Below we provide protocols for 1-ml and 200-µl assays. If you have access to a spectrophotometer
equipped to read microtiter plates, you may find it more convenient to use the 200-µl assay
protocol, which is intended for use with 96-well, flat-bottom microtiter plates.
Note: The experimental conditions and volumes of reagents used throughout the α-Galactosidase Quanitative Assay
have been carefully tested and optimized for use in the 1-ml and 200-µl assay formats described below. Please
follow as directed. Though we do not recommend changing the actual volumes of reagents used in the colorimetric
assay, if the signal from an experimental sample exceeds the linear range of the assay, you can dilute the media
supernatant before transferring an aliquot to the reaction tube or well. Remember to correct for individual sample
volumes and dilutions when tabulating final results at Step 11.
5.Prepare a sufficient amount of Assay Buffer for all samples including controls, and allow
to equilibrate to room temperature.
www.clontech.com
• For each 1-ml assay, you will need 24 µl Assay Buffer.
• For each 200-µl assay (96-well microtiter plate format), you will need 48 µl Assay
Buffer.
Note: We recommend assaying each sample in triplicate. Be sure to include positive and negative controls in
your assay. Also include a reagent blank in each assay to calibrate the spectrophotometer prior to reading the
OD of your samples. A reagent blank is composed of sterile, unused culture media, Assay Buffer, and Stop
Solution combined according to Steps 6–9.
Assay Scale
200-µl
1-ml
6.Transfer cell culture medium supernatant (from Step 4)16µl
8µl
into a 1.5-ml microcentrifuge tube or into a well of a
clear microtiter platea.
7.Add Assay Buffer to each sample.
48µl
24µl
8.Incubate at 30°C for 60 min. Be sure to cover
microtiter plates with a lid or parafilm to prevent
evaporation.
9.Terminate the reaction by adding Stop Solution.
136 µl of 10X
960 µl of 1X
96-well plate
1.5-ml cuvette
Note: Use 1X Stop Solution for 1-ml assays and
10X Stop Solution for 200-µl assays.
10.Record the optical density of each sample at 410 nm (OD410)b.
a
b
Add a corresponding volume of sterile, unused culture media to the reagent blank.
Zero the spectrophotometer using the reagent blank and measure the OD410 of the experimental and control
samples relative to the blank.
11.Calculate α-galactosidase units. One unit of α-galactosidase is defined as the amount
of enzyme that hydrolyzes 1 µmole p-nitrophenyl-α-d-galactoside to p-nitrophenol and
d-galactose in 1 min at 30°C in acetate buffer, pH 4.5 (Lazo et al., 1978).
α-galactosidase
[milliunits/(ml x cell)] =
OD410 x Vf x 1,000/[(ε x b) x t x Vi x OD600]
Vf = final volume of assay (200 µl or 992 µl)
Vi = volume of culture medium supernatant added (16 µl or 8 µl)
OD600 = optical density of overnight culturea
ε x b = p-nitrophenol molar absorbtivityb at 410 nm x the light path (cm)
= 10.5 (ml/µmol) for 200-µl formatb,c
= 16.9 (ml/µmol) for 1-ml format where b = 1 cmb,d
The optical density at 600 nm, recorded in Step 2, is used to normalize the OD410 of different media samples to
the number of cells in each culture.
b
The molar absorbtivity, though independent of concentration and light path, varies with the chemical properties
(e.g., pH) of the solution. Because different strengths and quantities of Stop Solution are used to terminate
the 200-and 1-ml reactions, the final pHs and, therefore, the molar absorbtivities are different for the two
formats.
c
Determined at Clontech using a SPECTRAmax ® Microplate Spectrophotometer and Corning Costar
UV-transparent, flat-bottom plates (Corning Cat No. 3635); Some microplate readers have pathlength correction
capabilities to normalize absorbance values to those obtained when the light pathlength is 1-cm (e.g., using
1.5-ml cuvettes). With pathlength correction on, the molar absorbtivity, ε, of p-nitrophenol at 410 nm in the
200-µl format was determined to be 20.3 ml/µmol.
The well-diameter and, therefore, the light path (b) in other brands of 96-well plates may differ from that of the
Corning plates used for these determinations. To determine ε x b in other plates, construct a plot of A410 versus
concentration of p-nitrophenol (PNP) as follows. Using a 10 µmol/ml standard solution of p-nitrophenol (Sigma
Cat No. 104-1), make 1:2 serial dilutions in water down to 0.02 µmol/ml PNP. Use these serial dilutions in place
of medium supernatant in Step 6, above. Then follow Steps 7, 9, and 10 as directed; omit Step 8. At Step 7, use
Assay Buffer that has been prepared by combining 2 volumes of 1X NaOAc with 1 volume of H2O, not PNPα-Gal. Finally, plot A410 versus concentration of PNP. According to the Beer-Lambert Law, the proportionality
constant, ε x b, is equal to the slope of the straight line defined by these data.
d
Determined at Clontech using 1.5-ml cuvettes
a
www.clontech.com
33
VII.Working With Yeast Plasmids
Isolating plasmid DNA from yeast is not trivial, primarily because of the tough cell wall.
Furthermore, the relatively large size (>6 kb) and low copy number (~50/cell) of some yeast
plasmids results in very low DNA yields, regardless of the plasmid isolation method used. In
addition, plasmid DNA isolated from yeast is often contaminated by genomic DNA because
yeast contain ~3X as much genomic DNA as E. coli, and the isolation method breaks the
yeast chromosomes and releases them from cellular material.
There are several yeast plasmid isolation procedures currently in use. The various protocols
differ primarily in the method used to break the cell walls. Here we provide the protocol that
we optimized for ourYeastmakerYeast Plasmid Isolation Kit (Cat No. 630441).This procedure,
which was modified from the method of Ling et al. (1995), uses extensive digestion with
lyticase to weaken the cell walls and SDS to burst the resulting spheroplasts. The DNA preps
can be cleaned up using either CHROMA SPINTM Columns or phenol:chloroform extraction
followed by ethanol precipitation. If CHROMA SPINTM Columns are used, this method takes
<2 hr from cell pellets to purified plasmid, and is simple enough to be easily adapted for
processing many samples simultaneously.
This purification method yields DNA of sufficient purity for use as a PCR template (Chapter
VIII) or for transforming E. coli (Chapter VII.C). However, if you need a large quantity of
plasmid, or very pure plasmid DNA, such as for sequencing or restriction enzyme digestion,
you will have to transform E. coli and prepare plasmid using standard methods (Sambrook
et al., 1989).
Plasmid rescue via complementation of E. coli mutations
Plasmid isolation from yeast cotransformants is complicated by the presence of two (or
more) types of plasmids in a single yeast colony. Nutritional selection of E. coli transformants
bearing the yeast plasmid of interest can be an efficient way to “rescue” one type of plasmid
from a mixture of plasmids bearing different nutritional transformation markers. For more
information on plasmid rescue via transformation of E. coli, see Section VII.C.
B.Plasmid Isolation from Yeast
Reagents and Materials Required
The Yeastmaker Yeast Plasmid Isolation Kit (Cat No. 630441) provides the SDS and lyticase solutions, CHROMA
SPIN-1000 DEPC-H2O Columns, and 2-ml centrifuge tubes for use with the columns.
• Appropriate SD liquid or agar medium to keep selection on the plasmids (Appendix C.A;
Appendix E).
• Sterile, 1.5-ml microcentrifuge tubes (or a 96-tube microtiter array, multichannel pipettors,
and centrifuge adaptor for multiwell plates).
• 20% SDS
• Lyticase Solution (5 units/µl in TE buffer; store at 4°C for up to 2 months or at –20°C for
up to 6 months. If colloidal material precipitates, mix the solution by inversion before
using.)
• Recommended: CHROMA SPIN-1000 DEPC-H2O Columns (Cat No. 636093) and 2-ml
centrifuge tubes for use with the columns
• If you do not use CHROMA SPIN Columns, you will need materials to perform
phenol:chloroform extraction and ethanol precipitation:
• Phenol:chloroform:isoamyl alcohol (25:24:1; See Sambrook et al., 1989, for information
on preparing neutralized phenol solutions)
• 10 M ammonium acetate
• 95–100% ethanol
www.clontech.com
VII.Working With Yeast Plasmids continued
1.Prepare yeast cultures for lysis (Step a, b, or c below).
a. From a solid patch of growth:
i. Spread a thin film of yeast cells (~2-cm2 patch) onto the appropriate SD agar
medium.
ii. Incubate plate at 30°C for 3–4 days. (The patch should show abundant yeast
growth.)
iii. Scrape up a portion of the patch (~10 mm2) and resuspend the cells in 50 µl of
sterile H2O or TE in a 1.5-ml microcentrifuge tube.
b. From a liquid culture:
i. Inoculate a large (2–4-mm), fresh (2–4-day-old) yeast colony into 0.5 ml of the
appropriate SD liquid medium. Vortex tube vigorously to completely break up
the colony and resuspend the cells.
ii. Incubate at 30°C overnight with shaking at 230–250 rpm.
iii. Spin down the cells by centrifuging at 14,000 rpm for 5 min.
iv. Carefully pour off the supernatant and resuspend pellets in the residual liquid
(total volume ~50 µl).
c. For semi-automated handling of a large number of samples:
i. Place a large (2–4-mm), fresh (2–4-day-old) yeast colony into 0.5 ml of the appropriate
SD liquid medium in separate wells of a 96-tube microtiter array. Vortex each tube
vigorously to resuspend the cells. (Alternatively, use 0.5 ml of an overnight SD
liquid culture instead of a yeast colony.)
ii. Using a centrifuge adapted for multiwell plates, centrifuge the entire array at 1,000
x g for 5 min to pellet the cells.
iii. Carefully pour (or draw) off supernatants and resuspend pellets in the residual
medium (~50 µl) by vortexing or pipetting up and down.
2. Add 10 µl of lyticase solution to each tube.Thoroughly resuspend the cells by vortexing
or repeatedly pipetting up and down.
3. Incubate tubes at 37°C for 30–60 min with shaking at 200-250 rpm.
[Optional] Check a drop of the cell suspension under a phase contrast microscope (400X) for the progress
of cell lysis by adding a drop of 20% SDS to the side of the coverslip. As they come into contact with the
SDS, most cells should lose their refractile appearance and appear as “ghost-like” spheroplasts. If there
are still many intact cells present, incubate the samples for another 30 min.
4. Add 10 µl of 20% SDS to each tube and vortex vigorously for 1 min to mix.
5. Put the samples through one freeze/thaw cycle (at –20°C) and vortex again to ensure
complete lysis of the cells.
6. If necessary, samples can be stored frozen at –20°C. If samples have been frozen,
vortex them again before using them.
7. Pour the entire contents of the tube from Step 5 above onto a prespun CHROMA
SPIN-1000 Column and purify the plasmid DNA according to the CHROMA SPIN User
Manual. Purified plasmid DNA will elute from the column.
If you do not use CHROMA SPIN Columns, clean up the prep as follows:
a.
Bring the volume of the sample up to 200 µl in TE buffer (pH 7.0).
b.
Add 200 µl of phenol:chloroform:isoamyl alcohol (25:24:1).
c.
Vortex at highest speed for 5 min.
d.
Centrifuge at 14,000 rpm for 10 min.
e.
Transfer the aqueous (upper) phase to a fresh tube.
f.
Add 8 µl of 10 M ammonium acetate and 500 µl of 95–100% Ethanol.
h.
Place at –70°C or in a dry-ice/ethanol bath for 1 hr.
i.
Centrifuge at 14,000 rpm for 10 min.
j.
Discard supernatant and dry the pellet.
k.
Resuspend pellet in 20 µl of H2O.
www.clontech.com
35
VII. Working With Yeast Plasmids continued
Note: The amount of plasmid DNA recovered is small relative to the contaminating genomic
DNA; therefore, it cannot be measured by A260 or seen on an agarose gel.
C. Transforming E. coli with Yeast Plasmids
We recommend using electroporation (Section C.1) when transforming E. coli with plasmids
isolated from yeast because of the relatively high transformation efficiency that can be
obtained. This is important because of the yeast genomic DNA that is present in yeastisolated plasmids; the presence of genomic DNA reduces the transformation efficiency of
the plasmids. However, if you choose to use chemically competent cells (Section C.2), it is
essential that the cells be able to yield a transformation efficiency of at least 107 cfu/µg (of
pUC19 DNA).
Nutritional selection of E. coli transformants
In the Matchmaker two-hybrid systems, cloning vectors carrying HIS3, LEU2 , or TRP1 markers
can be selectively rescued by complementation of the E. coli hisB, leuB, or trpC mutations,
respectively. (The yeast HIS3, LEU2 , and TRP1 genes are expressed well enough in E. coli
to allow this complementation.) Furthermore, due to incompatibility of the E. coli plasmid
replication origins used on the different vectors, only one plasmid construct will propagate
in a given E. coli transformant plated on selection medium. Thus, there is no need to screen
every E. coli transformant for the presence of the other (unwanted) plasmids.
If you plan to perform a nutritional selection for plasmid rescue, we recommend using E. coli
srain KC8, which carries the hisB, leuB, and trpC mutations (K. Struhl, personal communication).
KC8 Chemically Competent (Cat No. 630434) and Electrocompetent (Cat No. 630435) Cells
are available from Clontech. HB101, which carries the leuB mutation (Bolivar & Backman,
1979), may be used to select for yeast plasmids bearing the LEU2 marker only.
For nutritional selection of KC8 and HB101 transformants on M9 minimal medium, add a
1X mixture of amino acids (i.e., dropout [DO] supplement) lacking the specific nutrient that
will allow selection of the desired plasmid (Appendix E). (The same DO supplements used
for yeast SD medium can be used to supplement M9 minimal medium; see Appendix C for
recipes).
• Because of its auxotrophic mutations, KC8 requires His, Leu,Trp, and thiamine for growth on
minimal medium, unless one of these nutrients is specifically omitted for the selection.
• HB101 requires leucine, proline, and thiamine for growth on minimal medium, unless one
of these nutrients is specifically omitted for the selection; note that HB101 is streptomycin
resistant.
• Although optional, we recommend including ampicillin (50 µg/ml) in the medium to reduce
background growth.
Any of the common E. coli host strains (e.g., DH5α; JM109) may be used if you prefer to select
transformants by resistance to ampicillin rather than using a nutrional selection. However,
because both the DNA-BD and AD plasmids will be represented in the E. coli transformant
population (and not necessarily in equal proportions), many transformant colonies will need
to be screened for the presence of the desired plasmid(s).The plasmids can be distinguished
by restriction enzyme digestion or PCR amplification using AD vector-specific insert-screening
primers.
Reagents and materials required
• E. coli competent cells (chemically competent or electrocompetent)
Notes:
• For methods to prepare electrocompetent E. coli cells, see Kaiser & Auer (1993), Dower et al. (1988), Chuang et al.
(1995), and Sambrook et al. (1989). Alternatively, purchase premade chemically competent or electrocompetent
E. coli cells from Clontech.
• If you use the direct electroporation method of Marcil & Higgins (1992), the E. coli competent cells must be
transformed at an efficiency of 109 cfu/µg (of pUC19 DNA) to work satisfactorily with yeast plasmids.
• For transformation of electrocompetent cells, you need an electroporator and a cuvette
with a 0.1-cm gap.
www.clontech.com
•
•
•
•
Yeast plasmid DNA (from Section B above)
Sterile, 14-ml polypropylene conical tubes (e.g., FalconTM Cat No. 2059)
Hanahan’s SOC medium or LB broth (Sambrook et al., 1989)
LB/amp (50 µg/ml) agar plates for antibiotic selection or appropriately supplemented M9/
amp plates for nutritional selection (Appendix C.B)
• Materials for isolating plasmid DNA from E. coli.
1.Procedure for transforming electrocompetent E. coli KC8
a. Prepare or thaw electrocompetent E. coli cells.
b. Add 1–2 µl of yeast plasmid solution to 40 µl of electrocompetent cells on ice.
c. Transfer samples to a prechilled cuvette having a 0.1-cm gap. Perform the electroporation
according to the manufacturer’s instructions.
d. Add 1 ml of LB or (preferably) SOC medium with no antibiotic to the cuvette.Transfer
the cell suspension to a 14-ml conical Falcon tube.
e. Incubate at 37°C for 1 hr with vigorous shaking (250 rpm).
e. Pellet cells by centrifuging at 2,500 rpm for 5 min in a tabletop centrifuge.
f. Discard supernatant and resuspend pellet in residual liquid.
g. Plate cells on supplemented M9/amp agar medium.
h. Incubate plates at 37°C for 24 hr (LB/amp selection only), or for 36–48 hr (for nutritional
selection on M9 medium). If you do not recover any colonies, see the Troubleshooting
tips below.
i. See Section C.3 for tips on plasmid isolation.
2.Procedure for transforming chemically competent E. coli KC8
Transformation efficiency is significantly affected by temperature.Therefore, prechill the
14-ml Falcon tubes and pipette tips to 4°C before using them.
a. Prepare the chemically competent cells or thaw them on ice.
b. Add 10 µl of yeast plasmid solution to a prechilled Falcon tube.
c. Add 100 µl of competent cells to the tube and mix well by gently tapping the tube.
d. Incubate on ice for 30 min.
e. Heat shock by transferring the tube to a 42°C water bath and incubating for 45–50
sec.
f. Chill on ice for 2 min.
g. Add 1 ml of LB broth or (preferably) SOC medium with no antibiotic.
h. Incubate at 37°C for 1 hr with vigorous shaking (250 rpm).
i. Pellet cells by centrifuging at 2,500 rpm for 5 min in a table-top centrifuge.
j. Discard supernatant and resuspend pellet in residual liquid.
k. Plate cells on appropriate medium (LB/amp or supplemented M9).
l. Incubate plates at 37°C for 24 hr (LB/amp selection only), or for 36–48 hr (for nutritional
selection on M9 medium). Typically, 10–100 colonies will be seen on the plate for a
successful transformation using plasmid isolated from yeast. If you do not recover
any colonies, see the Troubleshooting tips below.
m. If you performed a parallel transformation using the control pUC19 DNA, calculate
the transformation efficiency. (The competent cells should have been transformed
with an efficiency of ≥1 x 107 cfu/µg. See Section V.E.23 for a sample calculation.)
n. See Section C.3 for tips on plasmid isolation.
3.Tips on Isolating plasmid DNA from the E. coli transformants
a. Use a standard plasmid mini-prep procedure to isolate plasmid DNA from the E. coli
transformants (Sambrook et al., 1989).
www.clontech.com
37
Notes:
• If you are using an endA+ bacterial strain such as KC8 or HB101 as the host strain, extra care must be
taken when preparing plasmid DNA because of the presence of endonuclease A. (See Sambrook et al.
[1989]1:1.22–1.23.)
• Boiling lysis is not recommended for isolation of plasmids from endA+ bacteria.
• If you are using a commercial plasmid preparation kit, follow the manufacturer’s directions for host
strains that are endA+.
• If you plan to use the plasmid for sequencing or other applications requiring highly purified DNA, the
plasmid should be extracted with phenol:chloroform:isoamyl alcohol and precipitated with ethanol
before use. Alternatively, CHROMA SPINTM+TE-400 Columns (Cat No. K1323-1) may be used to purify
the plasmid.
b. To verify that you have obtained the correct plasmid, amplify the insert by PCR, digest
it with Alu I or Hae III, and run a small sample on an agarose/EtBr gel. Compare the
restriction digestion pattern with that of the original clone isolated from yeast.
4. Troubleshooting tips
a. If you do not obtain any transformants, you may need to improve the transformation
effiency of the cells.
• If you performed a nutritional selection on M9 minimal medium, repeat the
transformation, but plate the cells on LB/amp instead. (The recovery of new
transformants is generally better on on LB/amp than on M9 medium.)Then replica
plate the Ampr transformants to the appropriate M9 minimal medium for selection
of the desired plasmid and to verify that the undesired plasmid(s) have been lost.
Note that it takes somewhat longer to see colonies on M9 medium than on LB.
• If you are not already doing so, use electrotransformation rather than chemical
transformation; higher transformation efficiencies are usually obtained with
electroporation.
• Use competent cells that are known to be transformed with a very high efficiency.
(Both chemically competent and electrocompetent cells are available from
Clontech.)
b. If you try the measures recommended in Section 4.a above and still do not recover any
E. coli transformants the problem may be the plasmid preparation or the plasmid
itself.
• The yeast plasmid preparation may have no plasmid DNA in it. Check the medium
you used for the overnight cultures. It is important to use a medium that maintains
selection on the desired plasmid. The working stock plate used as your inoculum
source should also keep selection on the plasmid. When you repeat the plasmid
isolation procedure, be sure to include the freeze/thaw cycle at Step VII.B.5 to
ensure complete cell lysis.
• Check the concentration of total DNA in your plasmid prep using absorbance at 260
nm or by running a small sample (10 µl) on a gel. Although plasmid DNA makes up
only a small fraction of the total DNA, you can at least confirm that you have DNA
in your prep. The larger chromosomal DNA fragments should be visible on a 1%
agarose/EtBr gel. (The limit of detection with EtBr staining is ~4 ng [Sambrook et
al., 1989, Appendix E.5].)
• Even if you have a substantial amount of DNA in your prep, there is a remote
possibility that the plasmid of interest has integrated into the yeast chromosome
and therefore cannot replicate autonomously when introduced into E. coli. If
the plasmid’s insert can be amplified by PCR (Chapter VIII), it may be possible to
recover the insert by subcloning from the PCR product.
• The plasmid may encode a protein that is toxic to E. coli. Again, it may be possible
to recover the insert by subcloning the PCR-amplified fragment.
www.clontech.com
VIII. Analysis of Yeast Plasmid Inserts by PCR
Sometimes a two-hybrid library screening results in many, even hundreds, of positive
candidate clones. However, a few abundant insert sequences may account for the majority.
Sorting colonies into groups will eliminate duplicates bearing the same plasmid insert and
will save time in the subsequent analysis. The cDNA inserts from all plasmids encoding
candidate interacting proteins can be amplified by PCR and sorted into groups based on
restriction digestion patterns. After colonies have been sorted, a representative clone from
each group can be transferred to a new master plate for further analysis.
To ensure efficient amplification of all inserts, regardless of size, we strongly recommend
the use of long-distance (LD) PCR (Barnes, 1994; Cheng et al., 1994) with the Advantage®
2 Polymerase Mix (Cat No. 639201). The Advantage® 2 PCR Kit (Cat No. 639206) provides
a Advantage® 2 Polymerase Mix (which includes TaqStartTM Antibody), a 10X Advantage® 2
PCR Buffer, 50X dNTP, a positive control template, a mix of positive control primers, and a
complete User Manual.
Clontech offers PCR primers designed to amplify inserts cloned into Matchmaker TwoHybrid System vectors. The insert-screening amplimers hybridize to sequences flanking the
multiple cloning site (MCS) of the respective vectors. If you purchase Matchmaker LD-Insert
Screening Amplimers, we recommend that you use the LD-PCR protocol that accompanies
that product. However, LD-Insert Screening Amplimers can also be used in conventional
PCR using a single DNA polymerase to amplify inserts up to 3 kb (e.g., Ausubel et al., [1995]
Chapters 15.1 & 15.3).
• Matchmaker AD LD-Insert Screening Amplimers (Cat No. 630433) are for amplifying inserts
in the GAL4 AD cloning vectors pGAD10, pGAD424, pGAD GL, pGAD GH, pGADT7, pACT,
and pACT2.
• Matchmaker pB42AD LD-Insert Screening Amplimers (Cat No. 9108-1) are for amplifying
inserts in the LexA system AD cloning vector pB42AD.
• Matchmaker DNA-BDVector Insert Screening Amplimers (Cat No. 5417-1) are for conventional
PCR amplification of inserts in the GAL4 DNA-BD cloning vectors pGBT9, pGBKT7, pAS2,
and pAS2-1.
• Matchmaker LexA DNA-BD Insert Screening Amplimers (Cat No. 9109-1) are for conventional
PCR amplification of inserts in pLexA and pGilda.
B. Tips For Successful PCR of Yeast Plasmid Templates
1.Optimization of thermal cycling parameters
The optimal cycling parameters will vary with different templates, primers, experimental
protocols, tubes, and thermal cyclers. Refer to the LD-Insert Screening Amplimers
User Manual, Ausubel et al. (1995), or Roux (1995) for suggestions on optimizing PCR
conditions. In some cases, “touchdown” PCR may be needed. We have found that
touchdown PCR significantly improves the specificity of many PCR reactions in a wide
variety of applications (Don et al., 1991; Roux, 1995). Briefly, touchdown PCR involves
using an annealing/extension temperature that is several degrees (typically 3–10°C) higher
than the Tm of the primers during the initial PCR cycles (typically 5–10). The annealing/
extension temperature is then reduced to the primer Tm for the remaining PCR cycles.
The change can be performed either in a single step or in increments over several cycles;
for example, use 72°C for the first five cycles, 70°C for the next 5 cycles, and 68°C for the
remaining cycles.
2.Primer design
Primer design is the single largest variable in PCR applications and the single most
critical factor in determining the success or failure of PCR reactions. For best results,
we recommend that you use LD-Insert-Screening Amplimers from Clontech. However,
if you design your own primers, be sure to use sequences flanking the MCS. Always
check and recheck your primer design before constructing or ordering primers.
Length and G/C content: In general, primers should have a Tm of at least 70°C to achieve
www.clontech.com
39
VIII. Analysis of Yeast Plasmid Inserts by PCR continued
optimal results in a two-step cycling program with a 68°C annealing/extension step.
Therefore, whenever possible, primers should be at least 22 nucleotides (nt) long
(25–30-mers are preferred) and should have a GC content of 45–60%.
3.Thermostable polymerase
The Advantage® 2 Polymerase Mixes are designed for LD PCR—i.e., they contain both
primary and proofreading polymerases to permit amplification of virtually any insert,
regardless of size. If you do not use an Advantage Polymerase Mix, you will need to
prepare your own polymerase mix from commercially available, LD PCR-licensed DNA
polymerases, such as Taq or AmpliTaq®. We also strongly recommend that you include
TaqStart Antibody in the polymerase mix, for automatic hot start (see Section B.4 below).
(TaqStart Antibody is premixed in the Advantage 2 Polymerase Mix.)
4.Use of antibody-mediated or conventional hot start
To minimize nonspecific amplification, we strongly recommend that you perform hot start
PCR.There are several methods available for hot start PCR, including those using wax beads
(Chou et al., 1992) or a manual hot start (D’Aquila et al., 1991). TaqStart Antibody
(Cat No. 639250, 639251) provides an automatic hot start when used with Taq or KlenTaq
DNA Polymerase (Kellogg et al., 1994). Antibody-mediated hot start withTaqStart Antibody
is more convenient than manual hot start or wax-bead-mediated hot start, and has been
proven to be at least as effective as the conventional methods.
5.Template quality
a. Because of the exponential nature of PCR amplification, many conventional PCR
applications such as screening cDNA inserts work well with templates of average
or even low quality, including plasmid DNA isolated from yeast. Use 1-2 µl of yeast
plasmid DNA preparation (from Section VII.B) per PCR.
b. Be sure to use a single, well-isolated yeast colony when inoculating liquid cultures
for preparation of plasmid from yeast (Chapter VII.B).
c. If the yeast transformant contains more than one plasmid insert sequence,
you may see multiple PCR bands. Restreak the yeast transformant on the
appropriate SD medium that maintains selection on the desired plasmid(s) but
not on their interactions (Appendix E). The extra generations of growth will allow
segregation (i.e., loss) of some of the plasmids. After reconfirming the presence
of positive plasmids using a β-gal colony-lift assay, repeat the plasmid isolation
and PCR analysis on well-isolated colonies. In some cases, it may be necessary
to transform E. coli with the yeast plasmid prep, and isolate plasmid from
E. coli transformants to ensure a homogeneous plasmid preparation (Chapter
VII.C).
6.Tips for characterizing PCR products
a. Electrophorese 10 µl samples of the PCR product on an EtBr/0.8% agarose gel to
confirm that the PCR worked and to determine if the plasmid prep contains multiple
(nonhomogeneous) plasmids.
b. Digest another 10-µl sample of each amplified insert with a frequent-cutter restriction
enzyme, such as Alu I or Hae III, in a 20-µl volume reaction. Run these samples on
an EtBr/1.8% agarose gel in parallel with DNA size markers for comparison.
7.Good PCR practices
a. Prepare reactions with dedicated pipettors in a dedicated work space
Due to the tremendous amplification power of PCR, minute amounts of contaminating
DNA can produce nonspecific amplification; in some instances, contaminants can
cause DNA bands even in the absence of added template DNA. We recommend that
you set up your PCR reactions in a dedicated lab area or noncirculating containment
hood and use dedicated pipettors, PCR pipette tips with hydrophobic filters, and
dedicated solutions. Perform post-PCR analysis in a separate area with a separate
set of pipettors.
www.clontech.com
VIII. Analysis of Yeast Plasmid Inserts by PCR continued
b. Pipetting
Because of the small volumes used in PCR experiments and the potential for tubeto-tube variation, careful pipetting technique is extremely important. Always be sure
that no extra solution is on the outside of the pipette tip before transfer. When adding
solution to a tube, immerse the tip into the reaction mixture, deliver the solution,
and rinse the pipette tip by pipetting up and down several times.
c. Use a Master Mix
To reduce tube-to-tube variation, use a master mix whenever you set up multiple PCR
reactions. If you wish, include the primers in the master mix also. If you are setting
up several sets of parallel samples, assemble multiple master mixes (e.g., each with
a different set of primers). The master mix should be thoroughly mixed before use
(i.e., vortexed without bubbling).
d. Always include positive and negative controls (i.e., H2O instead of DNA template).
Positive controls are provided with all of Clontech’s Insert Screening Amplimer
Sets.
www.clontech.com
41
IX. Additional Useful Protocols
A. Yeast Colony Hybridization
Yeast colony hybridization is an efficient way to screen a large collection of library transformants
for the presence of an abundant cDNA insert. Duplicate colonies bearing the same library plasmid
can then be eliminated from further analysis. We have had success with this modification of
the classic protocol of Grunstein and Hogness (1975; Kaiser, et al., 1994; Ausubel et al., 1994).
In this procedure, colonies are directly lifted onto a nylon membrane. β-glucuronidase is used
to break cell walls.
Reagents and Materials Required
• Appropriate SD agar plates that will keep selection on the plasmid(s) of interest (Appendix
E or the system-specific User Manual)
• Labeled cDNA probe complementary to previously isolated cDNAs
Note: Oligonucleotides, random-primed cDNAs, or PCR-generated fragments can be used as probes.
Oligonucleotide probes may be advantageous if the cDNA is a member of a protein family to avoid inadvertently
excluding related genes that are not identical to those initially obtained.
• 1 M sorbitol/20mM EDTA/50 mM DTT (prepare fresh)
• 1 M sorbitol/20 mM EDTA
• 0.5 M NaOH
• 0.5 M Tris-HCl (pH 7.5)/6X SSC (Ausubel et al., 1994)
• 2X SSC (Ausubel et al., 1994)
• 100,000 units/ml β-glucuronidase (type HP-2 crude solution from Helix pomatia; Sigma Cat No. G-7017)
• 82-mm circular nylon membrane, sterile
• Whatman 3 MM paper
• 80°C vacuum oven or UV cross-linker
• Additional reagents and equipment for bacterial filter hybridization (Ausubel et al., 1994)
1.If you have not done so already, collect the colonies to be screened onto a master plate
in a grid pattern to facilitate future identification of the colonies. Include a positive and
negative control on each plate. Since this will be your master plate, it is important to
use the appropriate SD agar medium to maintain selection on all plasmids (including
any reporter plasmid). Incubate plate at 30°C for 2–4 days until colonies appear.
2.Prepare sorbitol/EDTA/DTT solution.
3.For each plate of colonies to be screened, presoak a Whatman 3 MM paper in the sorbitol/
EDTA/DTT solution.
4. Using forceps, place a sterile, prelabeled, dry nylon membrane over the surface
of the plate of colonies to be assayed. Gently rub the membrane with the side of the
forceps to help colonies cling to the membrane.
5.Poke holes through the membrane into the agar in three or more assymetric locations
to orient the membrane to the master plate.
6. When the membrane has been evenly wetted, carefully lift it off the agar plate
with forceps and allow it to air dry briefly (~5 min). Place membrane, colony-side-up, on
a presoaked sheet of Whatman 3 MM paper (from Step 3 above) and incubate for ~30
min.
7.Optional: Place membranes at –70°C for 5 min, then thaw at room temperature for one
or more cycles to facilitate the disruption of the cell walls.
8. Dilute the β-glucuronidase 1:500 in sorbitol/EDTA. Use 2 µl (of the 100,000 units/ml
β−glucuronidase stock) per ml of sorbitol/EDTA to give a final concentration of 200 units/
ml). Allow 3–5 ml of diluted β-glucuronidase per filter to be screened.
9.For each membrane to be screened, cut another piece of Whatman 3 MM paper to
fit inside a 100-mm petri dish. Place the paper disc in the dish containing the diluted
β-glucuronidase to saturate the paper. Remove excess liquid.
10.Carefully layer the nylon membrane, colony side up, on top of the β-glucuronidase-soaked
filter. Avoid trapping air bubbles in between the two layers. Cover the dish. Incubate the
membrane on the filter for up to 6 hr at 37°C until >80% of the cells lack a cell wall.
www.clontech.com
IX. Additional Useful Protocols continued
Note: The extent of cell wall removal can be determined by removing a small quantity of cells from the filter
to a drop of sorbitol/EDTA on a microscope slide, and observing directly with a phase-contrast microscope at
≥60X magnification. Cells lacking a cell wall are nonrefractile.
11. Place membrane on Whatman 3 MM paper saturated with 0.5 M NaOH for 8–10
min.
12. Place membrane on Whatman 3 MM paper saturated with 0.5 M Tris-HCl (pH
7.5)/6X SSC for 5 min. Repeat step 12 with a second sheet of presoaked Whatman 3 MM
paper.
13. Place membrane on Whatman 3 MM paper saturated with 2X SSC for 5 min. Then
place membrane on dry Whatman paper to air dry for 10 min.
14. Bake membrane at 80°C for 90 min in a vacuum oven or UV cross-link.
15. Proceed as for bacterial filter hybridization (Ausubel et al., 1994).
B. Generating Yeast Plasmid Segregants
For some applications, it is useful to generate a segregant strain that has only a single type of
plasmid from yeast cotransformants containing more than one kind of plasmid.There are several
ways this can be accomplished.The most reliable but also most time-consuming way is to isolate
the mixed plasmid DNA from yeast, use it to transform E. coli, isolate the desired plasmid from
E. coli transformants, and transform the desired yeast host strain with the isolated plasmid DNA.
Alternatively, the yeast cotransformant strain can be grown for several generations on SD medium
that maintains selection on the desired plasmid only, as described in Section B.1 below.The search
for yeast segregants can be significantly accelerated if you are working with a cycloheximideresistant yeast host strain and the unwanted plasmid confers sensitivity to cycloheximide,
as described in Section B.2 below. Cycloheximide counterselection is an option with the
Matchmaker Two-Hybrid System 2 (Cat No. K1604-1), but cannot be used with the host strains
provided with Pretransformed Matchmaker Libraries or the original Matchmaker System (Cat
No. K1605-1).
1.Segregation by natural loss of an unselected plasmid
a. Culture individual cotransformant colonies (separately) in 3 ml of the appropriate SD liquid
selection medium for 1–2 days at 30°C with shaking (230–250 rpm). The medium must
maintain selection on the plasmid of interest, but not on the plasmid you wish to lose.
Under these conditions, the plasmids that are not selected for are lost at a rate of 10–20%
per generation. Refer to Appendix E for information on yeast plasmid transformation/
selection markers.
b. Spread a diluted sample of this liquid culture on agar plates that will select for the
desired plasmid.
www.clontech.com
43
c. Incubate the plate at 30°C for 2–3 days or until colonies appear.
d. Using sterile toothpicks or pipette tips, transfer 20–30 individual colonies (in an
orderly grid fashion) to appropriate SD selection plates to verify that they have lost
the unwanted plasmid and retained the plasmid of interest.
Note: Store the yeast segregants on the appropriate SD selection plates wrapped in Parafilm at 4°C for
up to two weeks.
2.Cycloheximide counterselection of yeast segregants
Some yeast host strains, such as CG-1945 and Y190, carry the cyhr2 mutant allele and
are cycloheximide resistant (CyhR; C. Giroux, personal communication, for CG-1945,
and Harper et al., 1993, for Y190). The wild-type CYHs2 gene is dominant to the cyhr2
mutant allele. Thus, when transformed with a plasmid such as pAS2-1 that contains the
wild-type CYHs2 gene, the host strain will become sensitive to cycloheximide; this holds
true for a CyhR host strain cotransformed with a CYHs2-bearing plasmid and another
plasmid that does not carry the CYHs2. gene. Therefore, one can effectively select for
yeast cells that have spontaneously lost the CYHs2-bearing plasmid while retaining the
other plasmid, simply by plating the cotransformants on the appropiate SD medium
containing cycloheximide.
Note: The CYH2 gene encodes the L29 protein of the yeast ribosome. Cycloheximide, a drug which blocks
polypeptide elongation during translation, prevents the growth of cells that contain the wild-type CYH2 gene.
Cycloheximide resistance results from a single amino acid change in the CYH2 protein. Cells containing both the
sensitive (wild-type) and the resistant (mutant) CYH2 alleles fail to grow on medium containing cycloheximide.
Therefore, the loss of a CYH2-containing plasmid can be selected for directly if the host carries the resistant
allele chromosomally (Guthrie & Fink [1991], pp 306–307).
a. From each of the restreaked (CyhS) cotransformants of interest, pick a colony, 1–3 mm
in diameter, and resuspend it in 200 µl of sterile H2O. Vortex thoroughly to disperse
the cells.
Note: Do not patch or streak cells from the colony over to the cycloheximide-containing medium. Cells
transferred in this way are at too high a density for the cycloheximide selection to work.
b. Spread 100 µl of the cell suspension onto an SD/–Leu/+cycloheximide plate. Also
spread 100 µl of a 1:100 dilution.
Note: The concentration of cycloheximide to use in the medium depends on the host strain. For example,
use 1.0 µg/ml for CG-1945; 10.0 µg/ml for Y190.
c. Incubate the plate at 30°C until individual CyhR colonies appear. (This usually takes 3–5
days.)
d. Transfer the CyhR colonies to appropriate SD selection plates to verify that they
have lost the CYHs2-bearing plasmid and retained the plasmid of interest. Refer to
Appendix E for information on yeast plasmid transformation/selection markers.
Note: These yeast clones are referred to as CyhR segregants. Store them on the appropriate SD selection
plates wrapped in Parafilm at 4°C for up to two weeks.
C. Yeast Mating
Yeast mating is a convenient method of introducing two different plasmids into the same
host cells, and, in some applications, can be used as a convenient alternative to yeast
cotransformations (Bendixen et al., 1994; Harper et al., 1993; Finley & Brent, 1994). See
Guthrie & Fink (1991) or Pringle et al. (1997) for information on the biology of yeast mating.
The following small-scale protocol works well for creating diploids by yeast mating. If you
wish to screen a Pretransformed Matchmaker Libary using yeast mating, please refer to the
User Manual provided with those libraries for an optimized, library-scale mating protocol.
1.Preparation for yeast mating
a. If you have not done so already, generate an appropriate yeast strain containing the
plasmid of interest.
b. Transform the chosen mating partner separately with the plasmids you wish to test
in combination with the plasmid of interest. Be sure to include transformations with
the appropriate negative and positive control plasmids, if applicable.
www.clontech.com
c. Select for transformants on the appropriate SD dropout medium.
d. For each plasmid of interest to be tested, set up pairwise yeast matings with
transformants containing control plasmids. Use either the standard procedure (Section
C.2) or the procedure adapted for microtiter (96-well) plates (Section C.3).
2.Yeast mating procedure (standard)
a. Pick one colony of each type to use in the mating. Use only large (2–3-mm), fresh
(<2-months old) colonies from the working stock plates.
b. Place both colonies in one 1.5-ml microcentrifuge tube containing 0.5 ml of YPD
medium. Vortex tubes to completely resuspend the cells.
c. Incubate at 30°C overnight (20–24 hr) with shaking at 200 rpm.
d. Spread 100-µl aliquots of the mating culture on the appropriate SD minimal media.
Use double dropout to select for both plasmids and triple dropout to select for diploids
in which a positive two-hybrid interaction is occurring. Proceed to step 4 below.
3.Yeast mating procedure (microtiter plate version)
If you have many plasmids of interest to mate to several control strains, it may be more
efficient to set up the matings in separate wells of a sterile, flat-bottom microtiter plate.
In between steps, keep plate covered with a sterile lid.
a. Aliquot 160 µl of YPD medium to each well.
b. For each plasmid of interest to be tested, place a single transformant colony in a 1.5-ml
microcentrifuge tube containing 1 ml of YPD. Vigorously vortex the tube to disperse the
cells.
c. For each type of control plasmid to be used, place several transformant colonies in
3 ml of YPD in a sterile, 10-ml conical tube. Vigorously vortex the tube to disperse
the cells.
d. Aliquot 20 µl of the cell suspension from Step 3.b into each well of a vertical column.
Use a separate column for each plasmid of interest to be tested.
e. Aliquot 20 µl of the cell suspension from Step 3.c into each well of a horizontal row.
Use a separate row for each type of control plasmid.
f. Place plate on a rotating platform shaker and incubate at 30°C for 6–18 hr at 200
rpm.
Note: Do not rotate at a higher speed or the medium will spill out of the wells.
g. Spread 100 µl of each mating culture on 100-mm plates containing the appropriate
SD minimal medium and proceed to next step.
4.Incubate plates at 30°C for 3–5 days to allow diploid cells to form visible colonies.
5.Score for growth on the SD agar plates.
6.Confirm nutritional and reporter phenotypes of diploids
To detect (or reconfirm) protein-protein interactions, assay the fresh diploid colonies
from the SD selection plates (Step 4) above for β-gal activity using the colony-lift filter
assay (Section VI.C). Discard any β-gal-positive colonies that contain the candidate library
plasmid alone.
www.clontech.com
45
X. References
Yeast Two-Hybrid System References:
Bartel, P. L., Chien, C.-T., Sternglanz, R. & Fields, S. (1993a) Using the two-hybrid system to detect protein-protein
interactions. In Cellular Interactions in Development: A Practical Approach., ed. Hartley, D.A. (Oxford University Press,
Oxford) pp 153–179.
Bartel, P. L, Chien, C.-T., Sternglanz, R. & Fields, S. (1993b) Elimination of false positives that arise in using the two-hybrid
system. BioTechniques 14:920–924.
Brent, R. & Ptashne, M. (1985) A eukaryotic transcriptional activator bearing the DNA specificity of a prokaryotic repressor.
Cell 43:729–736.
Chien, C. T., Bartel, P. L., Sternglanz, R. & Fields, S. (1991) The two-hybrid system: A method to identify and clone genes
for proteins that interact with a protein of interest. Proc. Nat. Acad. Sci. USA 88:9578–9582.
Fields, S. & Song, O. (1989) A novel genetic system to detect protein-protein interactions. Nature 340:245–247.
Fields, S. (1993)The two-hybrid system to detect protein-protein interactions. METHODS: A Companion to Meth. Enzymol.
5:116–124.
Fields, S. & Sternglanz, R. (1994) The two-hybrid system: an assay for protein-protein interactions. Trends Genet. 10:
286–292.
Fritz, C. C. & Green, M. R. (1992) Fishing for partners. Current Biol. 2:403–405.
Golemis, E. A., Gyuris, J. & Brent, R. (1996) Analysis of protein interactions; and Interaction trap/two-hybrid systems to identify
interacting proteins. In Current Protocols in Molecular Biology (John Wiley & Sons, Inc.), Chapters 20.0 and 20.1.
Golemis, E. A., Gyuris, J. & Brent, R. (1994) Interaction trap/two-hybrid systems to identify interacting proteins. In Current
Protocols in Molecular Biology (John Wiley & Sons, Inc.), Ch. 13. 14.
Guarente, L. (1993) Strategies for the identification of interacting proteins. Proc. Natl. Acad. Sci. USA 90:1639–1641.
Gyuris, J., Golemis, E., Chertkov, H. & Brent, R. (1993) Cdi1, a human G1 and S phase protein phosphatase that associates
with Cdk2. Cell 75:791–803.
Luban, J. & Goff, S. P. (1995) The yeast two-hybrid system for studying protein-protein interactions. Curr. Opinion in Biotechnol.
6:59–64.
Mendelsohn, A. R. & Brent, R. (1994) Biotechnology applications of interaction traps/two-hybrid systems. Curr. Opinion
in Biotechnology 5:482–486.
Other References:
Aho, S., Arffman, A., Pummi, T. & Uitto, J. (1997) A novel reporter gene MEL1 for the yeast two-hybrid system. Anal.
Biochem. 253: 270–272.
Alexandre, C., Grueneberg, D. A. & Gilman, M. Z.(1993) Studying Heterologous Transcription Factors in Yeast. METHODS:
A Companion to Methods in Enzymology 5:147–155.
Ammerer, G. (1983) Expression of Genes in Yeast Using the ADCI Promoter. Methods in Enzymology 101:192–201.
Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., Struhl, K. (1994) Current Protocols in
Molecular Biology (John Wiley & Sons, Inc.) Vol. 1, Chap. 5.
Barnes, W. M. (1994) PCR amplification of up to 35-kb DNA with high fidelity and high yield from λ bacteriophage templates.
Proc. Natl. Acad. Sci. USA 91:2216–2220.
Beier, D. R. & Young, E. T. (1982) Characterization of a regulatory region upstream of the ADR2 locus of S. cerevisiae.,
Nature 300:724–728.
Bendixen, C., Gangloff, S. & Rothstein, R. (1994) A yeast mating-selection scheme for detection of protein-protein
interactions. Nucleic Acids Res. 22:1778–1779.
Bolivar, F. & Backman, K. (1979) Plasmids of Escherichia coli as cloning vectors. Methods Enzymol. 68:245–267.
Breeden, L. & Nasmyth, K. (1985) Regulation of the Yeast HO Gene. Cold Spring Harbor Symposium Quant. Biol.
50:643–650.
Campbell, K. S., Buder, A. & Deuschle, U. (1995) Interactions between the amino-terminal domain of p56lck and cytoplasmic
domains of CD4 and CD8-alpha in yeast. Eur. J. Immunol. 25:2408-2412.
Campbell, K.S., Buder, A., Deuschle, U. (1995) Interaction of p56lck with CD4 in the yeast two-hybrid system. Ann NY Acad
Sci. 766:89-92.
Cheng, S., Fockler, C., Barnes, W. M. & Higuchi, R. (1994) Effective amplification of long targets from cloned inserts and
human genomic DNA. Proc. Natl. Acad. USA 91:5695–5699.
Chien, C. T., Bartel, P. L., Sternglanz, R. & Fields, S. (1991) The two-hybrid system: A method to identify and clone genes
for proteins that interact with a protein of interest. Proc. Nat. Acad. Sci. USA, 88:9578–9582.
Chou, Q., Russell, M., Birch, D., Raymond, J. & Bloch, W. (1992) Prevention of pre-PCR mispriming and primer dimerization
improves low-copy-number amplifications. Nucleic Acid Res. 20:1717–1723.
www.clontech.com
X. References continued
Chuang, S.-E., Chen, A.-L. & Chao, C.-C. (1995) Growth of E. coli at low temperature dramatically increases the transformation
frequency by electroporation. Nucleic Acids Res. 23:1641.
D’Aquila, R.T., Bechtel, L. J., Videler, J. A., Eron, J. J., Gorczyca, P. & Kaplan, J. C. (1991) Maximizing sensitivity and specificity
of PCR by preamplificiation heating. Nucleic Acids Res. 19:3749.
Don, R. H., Cox, P. T., Wainwright, B. J., Baker, K. & Mattick, J. S. (1991) ‘Touchdown’ PCR to circumvent spurious priming
during gene amplification. Nucleic Acids Res. 19:4008.
Dower, W. J., Miller, J. F. & Ragsdale, W. W. (1988) High efficiency transformation of E. coli by high voltage electroporation.
Nucleic Acids Res. 16:6127–6145.
Durfee, T., Becherer, K., Chen, P. L., Yeh, S. H., Yang, Y., Kilbburn, A. E., Lee, W. H. & Elledge, S. J. (1993) The retinoblastoma
protein associates with the protein phosphatase type 1 catalytic subunit. Genes Devel. 7:555–569.
Ebina, Y., Takahara, Y., Kishi, F. & Nakazawa, A. (1983) LexA protein is a repressor of the colicin E1 gene. J. Biol. Chem.
258:13258–13261.
Estojak, J., Brent, R. & Golemis, E. A. (1995) Correlation of two-hybrid affinity data with in vitro measurements. Mol. Cell
Biol. 15:5820–5829.
Feilotter, H. E., Hannon, G. J., Ruddel, C. J. & Beach, D. (1994) Construction of an improved host strain for two hybrid
screening. Nucleic Acids Res. 22:1502–1503.
Finley, Jr., R. L. & Brent, R. (1994) Interaction mating reveals binary and ternary connections between Drosophila cell cycle
regulators. Proc. Natl. Acad. Sci. USA 91:12980–12984.
Flick, J. S. & Johnston, M. (1990) Two systems of glucose repression of the GAL1 promoter in Saccharomyces cerevisiae.
Mol. Cell. Biol.10(9):4757–4769.
Gietz, D., St. Jean, A., Woods, R. A., & Schiestl, R. H. (1992) Improved method for high efficiency transformation of intact
yeast cells. Nucleic Acids Res. 20:1425.
Gimeno, R. E., Espenshade, P., & Kaiser, C. A. (1996) COPII coat subunit interactions: Sec24p and Sec23p bind to adjacent
regions of Sec16p. Mol. Biol. Cell 7:1815-23.
Giniger, E., Varnum, S. M. & Ptashne, M. (1985) Specific DNA binding GAL4, a positive regulatory protein of yeast. Cell 40:
767–74.
Giniger, E. & Ptashne, M. (1988) Cooperative DNA binding of the yeast transcriptional activator GAL4. Proc. Natl. Acad.
Sci. USA 85:382–386.
Golemis, E. A., Gyuris, J. & Brent, R. (1996) Analysis of protein interactions; and interaction trap/two-hybrid systems to
identify interacting proteins. In Current Protocols in Molecular Biology (John Wiley & Sons, Inc.), Ch. 20.0 and 20.1.
Guthrie, C. & Fink, G. R. (1991) Guide to yeast genetics and molecular biology. In Methods in Enzymology (Academic
Press, San Diego)194:1–932.
Harper, J. W., Adami, G. R., Wei, N., Keyomarsi, K. & Elledge, S. J. (1993) The p21 Cdk-interacting protein Cip1 is a potent
inhibitor of G1 cyclin-dependent kinases. Cell 75:805–816.
Heslot, H. & Gaillardin, C., eds. (1992) Molecular Biology and Genetic Engineering of Yeasts, CRC Press, Inc.
Hill, J., Donald, K. A. & Griffiths, D. E. (1991) DMSO-enhanced whole cell yeast transformation. Nucleic Acids Res.
19:5791.
Hope, I. A. & Struhl, K. (1986) Functional dissection of a eukaryotic transcriptional protein, GNN4 of yeast. Cell
46:885–894.
Ito, H., Fukada,Y., Murata, K. & Kimura, A. (1983)Transformation of intact yeast cells treated with alkali cations. J. Bacteriol.
153:163–168.
Iwabuchi, K., Li, B., Bartel, P. & Fields, S. (1993) Use of the two-hybrid system to identify the domain of p53 involved in
oligomerization. Oncogene 8:1693–1696.
Iyer, V. & Struhl, K. (1995) Mechanism of differential utilization of the his3 TR and TC TATA elements. Mol. Cell. Biol.
15:7059-7066.
James, P., Halladay, J. & Craig, E. A. (1996) Genomic libraries and a host strain designed for highly efficient two-hybrid
selection in yeast. Genetics 144:1425–1436.
Johnston, M., Flick, J. S. & Pexton, T. (1994) Multiple mechanisms provide rapid and stringent glucose repression of GAL
gene expression in Saccharomyces cerevisiae. Mol. Cell. Biol. 14:3834–3841.
Kaiser, P. & Auer, B. (1993) Rapid shuttle plasmid preparation from yeast cells by transfer to E. coli. BioTechniques
14:552.
Kellogg, D. E., Rybalkin, I., Chen, S., Mukhamedova, N., Vlasik, T., Seibert, P. & Chencik, A. (1994) TaqStart Antibody: Hot
start PCR facilitated by a neutrualizing monoclonal antibody directed against Taq DNA polymerase. BioTechniques
16:1134–1137.
Lazo, P. S., Ochoa, A. G. & Gascón, S. (1978) α-galactosidase (melibiase) from Saccharomyces Carlsbergenesis: Structural
and kinetic properties. Arch. Biochem. Biophys. 191:316–324.
www.clontech.com
47
X. References continued
Li, B. & Fields, S. (1993) Identification of mutations in p53 that affect its binding to SV40 T antigen by using the yeast
two-hybrid system. FASEB J. 7:957–963.
Li, L., Elledge, S. J., Peterson, C.A., Bales, E. S. & Legerski, R. J. (1994) Specific association between the human DNA repair
proteins XPA and ERCC1. Proc. Natl. Acad. Sci. USA 91:5012–5016.
Liljeström, P. L. (1985) The nucleotide sequence of the yeast MEL1 gene. Nucleic Acids Res. 13:7257–7268.
Ling, M., Merante, F. & Robinson, B. H. (1995) A rapid and reliable DNA preparation method for screening a large number
of yeast clones by polymerase chain reaction. Nucleic Acids Res. 23:4924–4925.
Liu, J., Wilson,T.E., Milbrandt, J. & Johnston, M. (1993) Identifying DNA-binding sites and analyzing DNA-binding domains
using a yeast selection system. In: Methods: A Companion to Methods in Enzymology 5:125–137.
Louret, O. F., Doignon, F. & Crouzet, M. (1997) Stable DNA binding yeast vector allowing high bait expression for use in
the two-hybrid system. BioTechniques 23: 816–819.
Luo, Y., Vijaychander, S., Stile, J. & Zhu, L. (1996) Cloning and analysis of DNA-binding proteins by yeast one-hybrid and
one-two-hybrid systems. BioTechniques 20:564–568.
Mahadevan, S. & Struhl, K. (1990) Tc, an unusual promoter element required for constitutive transcription of the yeast
HIS3 gene. Mol. Cell. Biol. 10:4447–55.
Marcil, R. & Higgins, D. R. (1992) Direct transfer of plasmid DNA from yeast to E. coli by electroporation. Nucleic Acids Res.
20:917.
Miller, J. H. (1972) Experiments in Molecular Genetics (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).
Miller, J. H. (1992) In A Short Course in Bacterial Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor; p.
74.
Post-Beittenmiller, M.A., Hamilton, R.W. & Hopper, J.E. (1984) Regulation of basal and induced levels of the MEL1 transcript
in Saccharomyces cerevisiae. Mol. Cell Biol. 4:1238–1245.
Pringle, J. R., Roach, J. R. & Jones, E. W., eds. (1997) The Molecular and Cellular Biology of the Yeast Saccharomyces: Cell
Cycle and Cell Biology (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).
Printen, J. A., & Sprague, G. F. Jr. (1994) Protein interactions in the yeast pheromone response pathway: Ste5p interacts
with all members of the MAP kinase cascade. Genetics 138:609-619.
Roux, K. H. (1995) Optimization and troubleshooting in PCR. PCR Methos Appl. 4:5185–5194.
Ruohonen, L. Aalto, M. K. & Keränen, S. (1995) Modifications to the ADH1 promoter of Saccharomyces cerevisiae for
efficient production of heterologous proteins. Journal of Biotechnology 39:193–203.
Ruohonen, L., Penttila, M. & Keränen, S. (1991) Optimization of Bacillus α-amylase Production by Saccharomyces cerevisiae.
Yeast 7:337–346.
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory,
Cold Spring Harbor, NY).
Sadowski, I., Niedbala, D., Wood, K. & Ptashne, M. (1991) GAL4 is phosphorylated as a consequence of transcriptional
activation. Proc. Natl. Acad. Sci. USA 88:10510-10514.
Schiestl, R. H. & Gietz, R. D. (1989) High efficiency transformation of intact cells using single stranded nucleic acids as a
carrier. Curr. Genet. 16:339–346.
Schneider, S., Buchert, M. & Hovens, C. M. (1996) An in vitro assay of β-galactosidase from yeast. BioTechniques
20:960–962.
Smirnov, M.N., Smirnov, V.N., Budowsky, E. I., Inge-Vechtomov, S.G. & Serebrjakov, N. G. (1967) Red pigment of adeninedeficient yeast Saccharomyces cerevisiae. Biochem. Biophys. Res. Commun. 27:299–304.
Stargell, L. A. & Struhl, K. (1996) Mechanisms of transcriptional activation in vivo: two steps forward. Trends Genet.
12(8):311-315.
Tirode, F., Malaguti, C., Romero, F., Attar, R., Camonis, J. & Egly, J. M. (1997) A conditionally expressed third partner stabilizes
or prevents the formation of a transcriptional activator in a three-hybrid system. J. Biol. Chem. 272: 22995–22999.
Tornow, J. & Santangelo, G. G. (1990) Efficient expression of Saccharomyces cerevisiae glycolytic gene ADH1 is dependent
upon a cis-acting regulatory element (UAS PRG) found initially in genes encoding ribosomal proteins. Gene 90:79–85.
van Aelst, L., Barr, M., Marcus, S., Polverino, A. & Wigler, M. (1993) Complex formation between RAS and RAF and other
protein kinases. Proc. Natl. Acad. Sci. USA 90:6213–6217.
Weisman, L. S., Bacallao, R. & Wickner, W. (1987) Multiple methods of visualizing the yeast vacuole permit evaluation of
its morphology and inheritance during the cell cycle. J. Cell Biol. 105:1539–1547.
West, R. W. Jr., Yoccum, R. R. & Ptashne, M. (1984) Saccharomyces cerevisiae GAL1-GAL10 Divergent Promoter Region:
Location and Function of the Upstream Activating Sequence UASG Molecular and Cellular Biology 4:2467–2478.
Yoccum, R. R. (1987) The GAL1, 7 and 10 upstream activator sequences are not enhancers, in Biological Research on
Industrial Yeasts. Vol. 3, Stewart, G., Russell, I., Klein, R. & Hiebsch, R., Ed., CRC Press, Boca Raton, Fl; page 61.
www.clontech.com
APPENDIX A. Glossary of Technical Terms
Note: Many of these terms have other meanings in different contexts. For brevity, we have included
only definitions relevant to this Yeast Protocols Handbook.
allele: One of two or more forms that can exist at a given genetic locus (e.g., his3-200 is a mutant
allele and HIS3 is a wild-type allele at the his3 locus). In standard yeast nomenclature, mutant
alleles are written in lower case italics, while wild-type alleles are written in upper case italics.
auxotroph: A strain of yeast or other microorganisms that will proliferate only when the medium
is supplemented with some specific nutrient not normally required by the organism. For example,
Trp– yeast strains are auxotrophic for tryptophan (Trp); they require Trp in the medium.
cis-acting element (or cis-acting locus): A DNA sequence that affects the transcriptional activity of
genes located on the same DNA molecule, often via binding of regulatory proteins or factors.
confluent: When yeast or bacterial colonies growing on an agar plate are so numerous that the
edges of the colonies touch each other.
clone: (a) A group of genetically identical cells or individuals derived by asexual division from a
common ancestor. (b) A heterologous cDNA fragment inserted into a vector; also refers to copies of
that original cDNA.
colony: A visible clone of cells growing on solid medium.
diploid: In yeast, a cell having two complete chromosome sets as a result of mating of haploid
a and α strains. A cell can also be diploid for one particular gene or several genes, due to the
presence of plasmids, or as a result of gene duplication.
dropout (DO) supplement: A mixture of several amino acids and nucleosides that must be added
to minimal synthetic medium to support the growth of yeast strains that have defined nutritional
requirements; typically, one or more specific nutrients is left (or “dropped”) out of the DO
supplement so that the resulting synthetic dropout (SD) medium will only support the growth of
yeast that are able to synthesize that nutrient.
gene: (a) The fundamental physical unit of heredity, recognized through its variant alleles; (b) a
DNA sequence that regulates and encodes a functional product, e.g., a polypeptide chain or an
RNA molecule.
genetic complementation: The production of a wild-type phenotype when (a) two different
mutations are combined in a diploid cell; or (b) when a wild-type allele on a plasmid is introduced
into a cell bearing a defective chromosomal allele via yeast mating or transformation.
genome: The entire complement of genetic material in a cell excluding autonomously replicating
plasmids and mitochondrial DNA.
genotype: Generally, a list of mutant alleles and exogenous genetic elements. Wild-type alleles
are sometimes listed as well for clarity in a specific experimental context.
haploid: A cell having one chromosome set. A diploid cell or organism can also be haploid for a
given gene due to chromosomal deletions.
hybridization probe: A defined nucleic acid segment which can be labeled and used to identify
specific DNA clones bearing the complementary sequence via hybridization.
leaky mutant: A mutant that represents a partial rather than a complete inactivation of the wildtype function; leaky phenotypes can result from a mutation in the coding region or in the promoter
region. In yeast one- and two-hybrid systems, some of the host strains are leaky for expression
of certain auxotrophic markers (for example, HIS3 expression in Y190).
mating types: A genetically haploid state of unicellular organisms that can reproduce sexually by
cellular and nuclear fusion to produce a diploid organism. In S. cerevisiae, there are two mating
types, a and α, which differ only physiologically and not in physical form.
mutant: An organism or cell carrying a mutation.
www.clontech.com
49
APPENDIX A. Glossary of Technical Terms continued
mutant allele: An allele differing from the allele found in the standard or wild type.
mutation: (a) The process that produces a gene or a chromosome differing from the wild type.
(b) The DNA or amino acid change resulting from such a process.
operator: In bacteria, a DNA region that acts a binding site for a specific repressor protein and
thereby exerts control over transcription of the adjacent structural gene or operon.
operon: In bacteria, a set of adjacent structural genes that are transcribed into a single mRNA
molecule, plus the adjacent regulatory genes that affect transcription of the structural genes.
PCR: Polymerase chain reaction; a process by which a defined segment of DNA is exponentially
replicated in vitro by the action of a thermostable DNA polymerase during repeated cycles of
heating and cooling.
phenotype: The observable properties of an organism determined by the organism’s genetic
constitution (genotype) and the effects of the environment.
plasmid: A genetic element in bacteria or yeast that can replicate autonomously in the host cell.
Some plasmids can also be inserted into the host’s genome in defined natural or experimental
situations, e.g., via transformation of linearized plasmid DNA.
promoter: A DNA sequence to which RNA polymerase complex binds and initiates transcription
of an adjacent structural gene or gene cluster. In yeast, the promoter is typically comprised of at
least one TATA box and other closely associated cis-regulatory elements (e.g., UASs).
prototroph: A strain of yeast or other microorganisms that will proliferate even if a particular
nutrient is not supplied in the medium. For example, Trp– yeast strains are protototrophic for Trp;
they can synthesize their own Trp from other biomolecules and do not require it in the medium. A
prototrophic transformation marker or reporter gene can be used to complement the corresponding
auxotrophic allele in another strain.
segregation: Genetically, the production from a single cell of two daughter cells having distinct
genotypes and phenotypes due to the separation of two alleles of a gene. In yeast, this can occur
during sporulation or in transformant clones as a result of loss of a plasmid.
trans-acting element: A gene that controls transcriptional activity of another gene through a
diffusable gene product (protein) such as a repressor or activator.
transformation: The process of introducing foreign DNA into a cell.
transformation markers: Genetic alleles whose phenotypes identify the presence of a plasmid
introduced into a cell; typically, such markers are genes that complement a nutritional requirement
or confer resistance to an antibiotic.
UAS: Upstream Activating Sequence; yeast DNA sequences that control the initiation of transcription
of adjacent structural genes via binding of specific regulatory proteins. An example is the binding
of the yeast GAL4 transcriptional activator (or DNA-BD) to the UASG of the GAL1 promoter.
wild type: The genotype or phenotype of an organism as it is found in nature or in a standard
laboratory strain.
www.clontech.com
APPENDIX B. Yeast Genetic Markers Used in the Matchmaker Systems
table iv. selected yeast genes and their associated phenotypes
Allele
Wild type
Mutant
Phenotype of mutant
TRP1
trp1-901 Trp–
Requires tryptophan (Trp) in the medium to grow, i.e., is a
Trp
auxotroph
LEU2
leu2-3, 112
Leu–
Requires leucine (Leu) to grow, i.e., is a Leu auxotroph
HIS3
his3-200
His–
Requires histidine (His) to grow, i.e., is a His auxotroph
URA3
ura3-52
Ura Requires uracil (Ura) to grow, i.e., is a Ura auxotroph
LYS2
lys2-801
Lys–
Requires lysine (Lys) to grow, i.e., is a Lys auxotroph
ADE2
ade2-101
Ade Requires adenine (Ade) to grow; i.e., is an Ade auxotroph;in
addition, confers a pink or red colony color to colonies growing
on media low in adenine. The red pigment is apparently an
oxidized, polymerized derivative of 5-aminoimidazole ribotide
which accumulates in vacuoles (Smirnov et al., 1967; Weisman
et al., 1987).
GAL4
gal4-542
Gal– (or gal4Δ)
Deficient in regulation of galactose-metabolizing genes (Flick
& Johnston, 1990; Johnston et al., 1994)
GAL80
gal80-538
Gal– Deficient in regulation of galactose-metabolizing genes (GAL
genes are constitutively expressed)
CYHs2
cyhr2
Cyhr
Resistant to cycloheximide
–
table v.
Reporter
Gene
–
Matchmaker reporter genes and their phenotypes
Gene
Description
Positive
Phenotypea
Negative
Phenotypea
lacZ
Encodes β-galactosidase LacZ+
LacZ–
• Blue colony
• White colony
• β-gal activity above
• Undetectable or background background level of β-gal activity
HIS3
Confers His prototrophy
His+
• Grows on SD/–His His–
• Does not grow on SD/–Hisb
LEU2
Confers Leu prototrophy
Leu+
• Grows on SD/–Leu
Leu–
• Does not grow on SD/–Leu
ADE2
Confers Ade prototrophy
Ade+
• Grows on SD/–Ade
Ade–
• Does not grow on SD/–Ade
• Pink or red colony color when grown on medium (such as YPD) low in Ade
Relative levels of background expression and reporter gene induction are dependent on the promoter constructs controlling
them. See Chapter II for information on the promoters.
b
5–60 mM 3-AT may be required to suppress leaky HIS3 expression in certain host strains and transformants and to obtain
an accurate His– phenotype.
a
www.clontech.com
51
APPENDIX C. Media Recipes
A.YEAST MEDIA
• YPD medium
YPD Medium (Cat No. 630409) and YPD Agar Medium (Cat No. 630410) are available in powder
form from Clontech. Our YPD Medium is a blend of peptone, yeast extract, and dextrose in
optimal proportions for growth of most strains of Saccharomyces cerevisiae. See Chapter XI
for ordering information. If you purchase Clontech’sYPD media, prepare the medium according
to the instructions provided. Alternatively, prepare your own YPD mixture as follows: 20 g/L Difco peptone
10 g/L Yeast extract
20 g/L Agar (for plates only)
• [Optional] For adenine-supplemented YPD (YPDA), add 15 ml of a 0.2% adenine hemisulfate
solution per liter of medium (final concentration is 0.003%, in addition to the trace amount
of Ade that is naturally present in YPD). Adenine hemisulfate tolerates autoclaving.
Add H2O to 950 ml. Adjust the pH to 6.5 if necessary, then autoclave. Allow medium to cool
to ~ 55°C and then add dextrose (glucose) to 2% (50 ml of a sterile 40% stock solution). Adjust
the final volume to 1 L if necessary.
Note: If you add the sugar solution before autoclaving, autoclave at 121°C for 15 min; autoclaving at a higher temperature,
for a longer period of time, or repeatedly may cause the sugar solution to darken and will decrease the performance
of the medium. Note that YPD from Clontech already contains glucose.
• [Optional] For kanamycin-containing medium, prepareYPD orYPDA as above. After autoclaved
medium has cooled to 55°C, add 0.2–0.3 ml of 50 mg/ml kanamycin (final concentration
10–15 mg/L).
• SD medium
Minimal SD Base and Minimal SD Agar Base, either with dextrose (glucose), or galactose +
raffinose, are available from Clontech in powder form. (See Chapter XI for ordering information.)
If you purchase Clontech’s Minimal SD Base, prepare your SD/Dropout (DO) medium according to
the instructions provided. For example, to prepare SD/–Leu/–Trp agar, you will need to combine
SD Minimal Agar Base (Cat No. 630412) with –Leu/–Trp DO Supplement (Cat No. 630412).
Alternatively, you can purchase yeast nitrogen base from another supplier (e.g., Difco Cat No.
0919-15-3) and prepare SD/DO medium as follows:
6.7 g
Yeast nitrogen base without amino acids
20 g
Agar (for plates only)
850 ml
H2O
100 ml of the appropriate sterile 10X Dropout Solution
• Adjust the pH to 5.8 if necessary, and autoclave. Allow medium to cool to ~ 55°C before
adding 3-AT, cycloheximide, additional adenine, or X-gal (see below).
• Add the appropriate sterile carbon source, usually dextrose (glucose) to 2%, unless specified
otherwise for your application. Adjust the final volume to 1 L if necessary.
Notes:
• If you add the sugar solution before autoclaving, autoclave at 121°C for 15 min; autoclaving at a higher temperature,
for a longer period of time, or repeatedly may cause the sugar solution to darken and will decrease the performance
of the medium. Note that SD Minimal Base from Clontech already contains a carbon source.
• If you purchase galactose separately, it must be highly purified and contain <0.01% glucose.
• [Optional] For 3-AT-containing medium, add the appropriate amount of 1 M 3-AT stock
solution and swirl to mix well. The concentration of 3-AT used in the medium depends on
the yeast strain and, to some extent, on the presence of transforming plasmid(s). See your
system-specific User Manual for further information.
Notes:
• 3-AT is heat-labile and will be destroyed if added to medium hotter than 55°C.
• 3-AT, a competitive inhibitor of the yeast HIS3 protein (His3p), is used to inhibit low levels of His3p expressed in
a leaky manner in some reporter strains (Fields, 1993; Durfee et al., 1993).
www.clontech.com
APPENDIX C. Media Recipes continued
• [Optional] For cycloheximide-containing medium, add the appropriate amount of 1 mg/
ml cycloheximide stock solution and swirl to mix well. The concentration of cycloheximide
used in the medium depends on the yeast strain. See your system-specific User Manual for
further information.
Notes:
• Cycloheximide is heat-labile and will be destroyed if added to medium hotter than 55°C.
• Cycloheximide-containing medium is used for selection of yeast strains, such as Y190 and CG-1945, carrying the
cyhr 2 allele.
• [Optional] If you wish to add excess adenine to SD medium, add 15 ml of 0.2% adenine
hemisulfate solution per liter of medium.
• Pour plates and allow medium to harden at room temperature. Store plates inverted, in a
plastic sleeve at 4°C.
• SD/Gal/Raf/X-gal plates
Prepare SD medium as described above except use 725 ml of H2O and do not adjust the pH.
Autoclave, and cool to ~ 55°C. Then add:
Final concentration
To prepare 1 L of medium
Galactose 2%
50 ml of 40% stock
Raffinose 1%
25 ml of 40% stock
10X BU salts
1X
100 ml of 10X stock
X-Gal 80 mg/L
4 ml of 20 mg/ml
Pour plates and allow medium to harden at room temperature. Store plates inverted, in a plastic
sleeve, in the dark, at 4°C for up to two months. Adjust final volume to 1L if necessary.
Notes:
• Galactose must be highly purified and contain <0.01% glucose.
• If the medium is too hot (i.e., >55°C) when the salt solution is added, the salts will precipitate. Also, X-Gal is heat
labile and will be destroyed if added to hot medium.
• BU salts must be included in the medium to adjust the pH to ~7, which is closer to the optimal pH for β-galactosidase
activity, and to provide the phosphate necessary for the β-gal assay to work.
• As the plates age, salt crystals will form in the medium. These do not affect the performance of the medium or the
results of the β-galactosidase assay.
• If you are assaying for expression of a lacZ reporter gene in a system that requires expression of a protein from an
intact yeast GAL1 promoter (such as in the Matchmaker LexA Two-Hybrid System), you must use 2% galactose +
1% raffinose as the carbon sources instead of glucose. If you are not using Clontech’s SD/Gal/Raf Minimal Base, be
sure to obtain high-quality galactose that is not contaminated by glucose.
Stock solutions for use with SD Media
• 1 M 3-AT (3-amino-1,2,4-triazole; Sigma Cat No. A-8056); prepare in deionized H2O and filter
sterilize. Store at 4°C. Store plates containing 3-AT sleeved at 4°C for up to 2 months.
• 10X BU Salts
Dissolve the following components in 1 L (total) of H2O:
70 g
Na2HPO4• 7H2O
30 g
NaH2PO4
Adjust to pH 7, then autoclave and store at room temperature.
• Carbon sources, filter sterilized or autoclaved:
Note: Autoclave at 121°C for 15 min; autoclaving at a higher temperature, for a longer period of time, or repeatedly
may cause the sugar solution to darken and will decrease the performance of the medium.
•40% Dextrose (glucose) Store at 4°C.
•40% Galactose (for LexA Two-Hybrid System; D(+) Galactose, e.g., Sigma Cat No. G-0750) Store at
4°C.
•40% Raffinose (for LexA Two-Hybrid System) Store at 4°C.
• 1 mg/ml (1000X) CHX (Cycloheximide; Sigma Cat No. C-7698); prepare in deionized H2O, filter
sterilize, and aliquot. Store at 4°C for up to 2 months. Store plates containing CHX sleeved at
4°C for up to 1 month.
• 50 mg/ml kan (kanamycin); prepare in deionized H2O, filter sterilize, and aliquot. Store at –20°C
for up to 1 month. Store plates containing kan sleeved at 4°C for up to 1 month.
www.clontech.com
53
• X-gal (20 mg/ml in DMF) Dissolve 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside in
N,N-dimethylformamide. Store in the dark at –20°C.
• 10X Dropout (DO) Solution
A combination of a Minimal SD Base and a DO Supplement will produce a synthetic, defined minimal
medium lacking one or more specific nutrients.The specific nutrients omitted depends on the selection
medium desired.To prepare SD/–Leu/–Trp agar, for example, combine –Leu/–Trp DO Supplement
(Cat No. 630417) with Minimal SD Agar Base (Cat No. 630412). (With this naming convention
[e.g., SD/–Leu/–Trp], if a nutrient is not indicated as missing, it is assumed to be present in the
medium.) Many of the commonly used DO Supplements can be purchased from Clontech.
Instructions for preparing the corresponding SD/DO medium are printed on the products’
labels.
Alternatively, make your own dropout supplement by combining the nutrients listed below
at the concentrations indicated to prepare a 10X Dropout Solution. A 10X Dropout Solution
contains all but one or more of these nutrients. Note that serine, aspartic acid, and glutamic
acid are not included in this list because they make the medium too acidic and because yeast
can synthesize these amino acids endogenously. However, if you wish to select for yeast strain
AH109 using medium that lacks methionine (i.e., on SD/–Met), you should add aspartic acid to
the 10X DO Solution—a 10X solution of aspartic acid is 1000 mg/L.
10X dropout supplements may be autoclaved and stored at 4°C for up to 1 year.
Nutrient
L-Adenine hemisulfate salt
L-Arginine HCl
L-Histidine HCl monohydrate
L-Isoleucine
L-Leucine
L-Lysine HCl
L-Methionine
L-Phenylalanine
L-Threonine
L-Tryptophan
L-Tyrosine
L-Uracil
L-Valine
10X Concentration
200 mg/L
200 mg/L
200 mg/L
300 mg/L
1000 mg/L
300 mg/L
200 mg/L
500 mg/L
2000 mg/L
200 mg/L
300 mg/L
200 mg/L
1500 mg/L
Sigma Cat. No.
A-9126
A-5131
H-8125
I-2752
L-8000
L-5626
M-9625
P-2126
T-8625
T-0254
T-3754
U-0750
V-0500
Example: To make one liter of 10X –Leu/–Trp DO Solution, combine the following:
•
•
•
•
•
•
•
•
•
•
•
200mg
200mg
200mg
300mg
300mg
200mg
500mg
2000mg
300mg
200mg
1500mg
1L
adenine hemisulfate
arginine HCl
histidine HCl monohydrate
isoleucine
lysine HCl
methionine
phenylalanine
threonine
tyrosine
uracil
valine
Dissolve components in 1 L deionized H2O. Autoclave.
www.clontech.com
B. E. coli MEDIA
• Hanahan’s SOC Medium
Final concentration To Prepare One Liter
Bactotryptone
2% 20 g
Yeast extract
0.5% 5 g
NaCl
10 mM
10 ml of 1 M NaCl
KCl
2.5 mM
2.5 ml of 1 M KCl
MgCl2*
10 mM
10 ml of 1 M MgCl2• 6 H2O
MgSO4*
10 mM
10 ml of 1 M MgSO4 • 7H2O
Glucose*
20 mM
20 ml of 1 M glucose
Deionized H2O to 1 L
* Before adding MgCl2, MgSO4, and glucose stock solutions, separately filter sterilize them using a 0.2-µm filter.
Add the bactotryptone, yeast extract, and NaCl to 900 ml of deionized H2O; stir or shake until
solutes have dissolved. Add the KCl. Adjust the pH to 7 with 5 N NaOH (~0.2 ml). Adjust the
volume to 960 ml with deionized H2O and autoclave. Just before use, add filter-sterilized
MgCl2, MgSO4, and glucose.
• LB broth
Bacto-tryptone
10 g/L
Bacto-yeast extract
5 g/L
NaCl
5 g/L
Adjust pH to 7.0 with 5 N NaOH. Autoclave. Store broth at 22°C.
• LB/amp agar plates
Prepare LB broth (Sambrook et al., 1989) as above. Add agar (18 g/L), autoclave, and cool
to 50°C. Add ampicillin to 50 µg/ml. Pour plates and store at 4°C.
• M9 minimal medium for nutritional selection of E. coli transformants complemented by
the wild-type yeast gene. For optimal recovery of KC8 and HB101 transformants, add a 1X
mixture of amino acids (i.e., dropout [DO] supplement) lacking the specific nutrient that
will allow selection of the desired plasmid. (The same DO supplements used for yeast SD
medium can be used to supplement M9 minimal medium; see Appendix C.A for dropout
recipe or purchase premixed DO Supplements from Clontech.) In addition, KC8 requires
thiamine, and HB101 requires thiamine and proline, for growth on minimal medium.
Prepare 900 ml of M9 medium as directed in Sambrook et al. (1989). To prepare agar plates,
add agar (20 g/L) prior to autoclaving. After autoclaving, allow medium to cool to 55°C.
Then add the following:
• 1 ml of 50 mg/ml ampicillin stock
• 1 ml of 1.0 M thiamine-HCl stock
• 100 ml of an appropriate sterile 10X DO stock solution
In addition, for HB101 cells only:
• 4 ml of a 10 mg/ml stock of proline
• Stock solutions for use with M9 or LB media
Ampicillin (50 mg/ml in H2O). Store at 4°C no longer than 1 month.
Thiamine-HCl (1 M, filter-sterilized)
Proline (10 mg/ml, filter sterilized)
10X DO stock solution (Appendix C.A)
www.clontech.com
55
APPENDIX D. Solution Formulations
A. For Preparation of Protein Extracts
• Protease Inhibitor Solution (concentrated)
Always prepare solution fresh just before using. Place on ice to prechill.
Type of protease(s)
To prepare 688 µl:
inhibited:
Pepstatin A 0.1 mg/ml 66 µl of a 1 mg/ml stock solution* Carboxyl proteases
(Sigma Cat No. P4265)
in DMSO
Leupeptin 0.03 mM 2 µl of a 10.5 mM stock solution*
Some thiol and
(Sigma Cat No. L2884)
serine proteases
Benzamidine 145 mM
500 µl of a 200 mM stock solution* Trypsin, plasmin,
(Sigma Cat No. B6506)
and thrombin
Aprotinin 0.37 mg/ml 120 µl of a 2.1 mg/ml stock solution*Some serine
(Sigma Cat No. A6279)
proteases
*Store the individual stock solutions as directed on the labels and follow label precautions.
• PMSF (phenylmethyl-sulfonyl fluoride) stock solution [100X*]
Dissolve 0.1742 g PMSF (Sigma Cat No. P7626) in 10 ml isopropanol. Wrap tube in foil
and store at room temperature. PMSF primarily inhibits serine proteases.
*Although this is a 100X stock solution, the final concentration of PMSF is greater than 1X in some mixtures,
i.e., PMSF is used in excess.
Caution: PMSF is hazardous. Wear gloves. Handle with care and read label precautions.
• Glass Beads (425–600 µm; Sigma Cat No. G-8772)
For Urea/SDS Protein Extraction Method:
• Cracking buffer stock solution Urea 8 M SDS 5% w/v
Tris-HCl [pH6.8] 40 mM EDTA* 0.1 mM Bromophenol blue
0.4 mg/ml Deionized H2O
* EDTA primarily inhibits metalloproteases.
To prepare 100 ml:
48 g
5g
4 ml of a 1 M stock solution
20 µl of a 0.5 M stock solution
40 mg
To a final volume of 100 ml
• Cracking buffer (complete): The following recipe is sufficient for one protein extract. Scale up recipe as required. Prepare only the volume you need just before use.
Because PMSF has a short half-life (~7 min) in aqueous solutions, you may need to add additional aliquots of PMSF during the course of the procedure. The initial excess PMSF
in the Cracking buffer quickly degrades.
To prepare 1.13 ml of complete Cracking buffer:
Cracking buffer stock solution 1 ml (recipe above)
β-mercaptoethanol 10 µl
Protease inhibitor solution 70 µl, prechilled (recipe above)
PMSF
50 µl of 100X stock solution
www.clontech.com
APPENDIX D. Solution Formulations continued
For TCA Protein Extraction Method:
• 20% w/v TCA in H2O (Store at 4°C; See Sambrook et al. [1989] for tips on preparing TCA solutions.)
• TCA Buffer
Place on ice to prechill before use.
Add the protease inhibitor solution and pMSF immediately prior to use.
To prepare 10 ml of TCA buffer:
Tris-HCl (pH 8)
20 mM
200 µl of a 1 M stock solution
Ammonium acetate
50 mM
66.6 µl of a 7.5 M stock solution
EDTA
2 mM
40 µl of a 0.5 M stock solution
Deionized H2O
9.7 ml
Protease inhibitor solution50 µl/ml 500 µl, prechilled (recipe above)
PMSF
100 µl of 100X stock solution
• SDS/glycerol stock solution SDS
7.3% w/v
Glycerol
29.1% v/v
Tris-base
83.3 mM
Bromophenol blue
Deionized H2O
To prepare 12 ml:
3.5 ml of a 25% stock solution
3.5 ml of 100%
1.0 ml of a 1 M stock solution, not pH-adjusted
Spatula tip-full
To a final volume of 12 ml
• Tris/EDTA solution
Tris-base
200 mM
EDTA
20 mM
Deionized H2O
To prepare 10 ml:
2.0 ml of 1 M stock solution, not pH-adjusted
0.4 ml of a 0.5 M stock solution
7.6 ml
• TCA-Laemmli loading buffer
Prepare fresh just prior to use.
To prepare 1 ml:
SDS/glycerol stock solution 480 µl (Stock solution may need to be warmed to 60°C to reliquefy)
Tris/EDTA solution
400 µl (Recipe above)
β-mercaptoethanol
50 µl
PMSF 20 µl PMSF stock solution (100X)
Protease inhibitor solution 20 µl Prechilled (recipe above)
Deionized H2O
30 µl
www.clontech.com
57
B. For Transformation of Yeast
• Carrier DNA (10 mg/ml)
Sonicated, carrier DNA in solution can be purchased separately (Cat No. 630440; see
Chapter XI for ordering information), or can be prepared using a standard method
(Sambrook et al., 1989). Just prior to use, denature the carrier DNA by placing it in a
boiling water bath for 20 min and immediately cooling it on ice. Use only high-quality
carrier DNA; nicked calf thymus DNA is not recommended.
• PEG/LiAc solution (polyethylene glycol/lithium acetate)
Prepare fresh just prior to use.
Final Conc. To prepare 10 ml of solution
PEG 4000
40%
8 ml of 50% PEG
TE buffer
1X
1 ml of 10X TE
LiAc 1X
1 ml of 10X LiAc
• Stock solutions
50% PEG 3350 (Polyethylene glycol, avg. mol. wt. = 3,350; Sigma Cat No. P-3640) prepare
with sterile deionized H2O; if necessary, warm solution to 50°C to help the PEG go into
solution.
100% DMSO (Dimethyl sulfoxide; Sigma Cat No. D-8779)
10X TE buffer: 0.1 M Tris-HCl, 10 mM EDTA, pH 7.5. Autoclave.
10X LiAc: 1 M lithium acetate (Sigma Cat No. L-6883) Adjust to pH 7.5 with dilute acetic
acid and autoclave.
C. For β-galactosidase Filter Assays
• Z buffer Na2HPO4 • 7H2O
16.1 g/L
NaH2PO4 • H2O
5.50 g/L
KCl
0.75 g/L
MgSO4 • 7H2O
0.246g/L
Adjust to pH 7.0 and autoclave.Can be stored at room temperature for up to 1 year.
• X-gal stock solution
Dissolve 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-GAL; Cat No. 8060-1) in
N,N-dimethylformamide (DMF) at a concentration of 20 mg/ml. Store in the dark at
–20°C.
• Z buffer/X-gal solution
100 ml Z buffer
0.27 ml β-mercaptoethanol (β-ME; Sigma Cat No. M-6250)
1.67 ml X-gal stock solution
D. For Liquid β-galactosidase Assays with ONPG as Substrate
• Z buffer (see preceding section for recipe)
• Z buffer with β-mercaptoethanol
To 100 ml of Z buffer, add 0.27 ml of β-mercaptoethanol.
• ONPG (o-nitrophenyl β-D-galactopyranoside; Sigma Cat No. N-1127) 4 mg/ml in Z buffer. Adjust to pH 7.0 and mix well.
Notes:
• ONPG requires 1–2 hr to dissolve. • Prepare solution fresh before each use.
www.clontech.com
E. For Liquid β-galactosidase Assays with CPRG as Substrate
• Buffer 1
To prepare 100 ml of solution
HEPES
2.38 g
NaCl
0.9 g
L-Aspartate [hemi-Mg salt; Sigma Cat No. A-9506] 0.065 g
BSA 1.0 g Tween 20 50.0 µl
Dissolve the above components in 75 ml of deionized H2O. Adjust pH to 7.25–7.30, then
bring volume to 100 ml. Filter sterilize. Store at 4°C for up to 3 months.
• Buffer 2 (20 ml)
Dissolve 27.1 mg of CPRG in 20 ml of Buffer 1 (final concentration of CPRG is 2.23
mM). Filter sterilize. Store at 4°C in the dark for up to 3 months.
F. For α-Gal Quantitative Assays
• PNP-α-Gal Solution
100 mM (p-nitrophenyl α-d-Galactopyranoside; Sigma Cat No. N0877) in deionized H2O
For 10 ml, dissolve 301.3 mg of PNP-α-Gal in 10 ml of deionized H2O. Filter sterilize.
Notes:
• Prepare solution fresh before each use.
• Keep the p-nitrophenyl α-d-Galactopyranoside solid anhydrous. Store in a dessicator at –20°C.
• 10X Stop Solution
1 M Na2CO3 in deionized H2O (Sigma Cat No. S7795)
• 1X NaOAc
0.5 M sodium acetate, pH 4.5 (Sigma Cat No. S7545)
• Assay Buffer
Prepare Assay Buffer fresh, before each use, by combining 2 volumes 1X NaOAc
Buffer with 1 volume PNP-α-Gal Solution [2:1 (v/v) ratio]. Mix well.
www.clontech.com
59
www.clontech.com
60 GenBank
Accession No. Hind III
U30497
(4.6, 2.2, 0.9, 0.7)
Hind III
U89961
(3.4, 2.1, 0.6, 0.35) Hind III U13188
(5.9, 0.7)d
Hind III U07647
(5.9, 0.7)d
Hind III
in submission
(7.1, 0.5, 0.3)
Hind III not available
(6.1, 0.5, 0.3)
Hind III
in submission
(7.1, 0.8)
Hind III
in submission
(4.9, 1.5, 0.9)
Hind III U07646
(4.6, 0.9)
Hind III
in submission
(0.2, 6.3)
Hind III
U89960
(5.2, 4.8, 0.2)
Hind III
in submission
(5.6, 0.9)
Hind III
U29899
(7.3, 0.8)
EcoR I
not available
(3.0, 3.05, 1.6)
Diagnostic
R.E. Sites (kb)
Golemis et al., 1996
Gimeno et al., 1996
Gyuris et al., 1993
(pEG202)
Tirode et al., 1997
Clontech
Louret et al., 1997
Bartel et al., 1993a
Clontech
van Aelst et al., 1993
van Aelst et al., 1993
Gyuris et al., 1993
(pJG4-5)
Harper et al., 1993
Li et al., 1994;
Elledge, pers. comm. Durfee et al., 1993;
Elledge, pers. comm. References (Plasmid
name in reference)
a
Key to system abbreviations: GAL4 2H-2 = Matchmaker Two-Hybrid System 2 (Cat No. K1604-1); LexA 2H = Matchmaker LexA Two-Hybrid System (Cat No. K1609-1); GAL4 2H =
Matchmaker Two-Hybrid System (Cat No. K1605-1); GAL4 2H-3 = Matchmaker Two Hybrid System 3 (Cat No. 630303). MM 1H = Matchmaker One-Hybrid System (Cat No. K1603-1).
Some plasmids are also available separately.
b
Additional vector information, restriction maps, and multiple cloning site (MCS) sequences are provided in the Matchmaker GAL4 Two-Hybrid System Vectors Handbook, the
LexA Two-Hybrid System User Manual, and Vector Information Packets (provided with regular and Pretransformed Matchmaker Libraries).
c
pAS2-1 is a derivative of the plasmid described in this reference; the plasmid was modified at Clontech. The EcoR I site is unique in pAS2-1.
d
pGAD424 is linearized by digestion with Sal I; pGAD10 does not contain a Sal I site.
GAL4(1–147) DNA-BD,
–Trp
8.4 TRP1, ampr, CYHs2
acidic activator B42,
–Trp
6.45 TRP1, ampr, HA epitope tag
GAL4(768–881) AD,
–Leu
6.6 LEU2, ampr
GAL4(768–881) AD,
–Leu
6.6 LEU2, ampr
GAL4(768–881) AD,
–Leu
7.9 LEU2, ampr
GAL4(768–881) AD,
–Leu
6.9 LEU2, ampr
GAL4(768–881) AD,
–Leu
8.0
LEU2, ampr, HA epitope tag
–Trp
7.3
TRP1, kanr, c-Myc epitope tag
–Trp
5.5 TRP1, ampr
LexA(1–202), DNA-BD,
–His
6.6
HIS3, ampr
LexA(1–202), DNA-BD,
–His
10.2 HIS3, ampr
–Trp
6.5
TRP1, ampr
8.1 –Leu
GAL4 2H-2
GAL4(768–881) AD,
& MM Libraries LEU2, ampr, HA epitope tag
pACT2
GAL4 2H-2
pAS2-1c
pB42AD
LexA 2H
pGAD10
MM Libraries
pGAD424 GAL4 2H
pGAD GH MM Libraries
pGAD GL MM Libraries
pGADT7
GAL4 2H-3
pGBKT7
GAL4 2H-3
pGBT9
GAL4 2H
pGilda
LexA 2H
pLexA
LexA 2H
pBridge
GAL4 2H,
GAL4 2H-2, -3
7.65 GAL4(768–881) AD,
–Leu
LEU2, ampr, HA epitope tag
MM Libraries
Size
(kb)
Matchmaker two-hybrid system cloning vectors
Selection on
SD Medium
table vi.
pACT
Descriptionb
Vector
Systema
APPENDIX E. Plasmid Information
table vii.
Human lamin C(66–230) –Trp
~9.0 in pAS2-1 TRP1, ampr
pLAM5’-1
GAL4 2H-2
www.clontech.com
SV40 large T-antigen (84–708) –Leu
~15.0 in pGAD3F, LEU2, ampr
in pACT2, LEU2, ampr
murine p53 (72–390) in pGBT9
–Trp
6.4 TRP1, ampr
murine p53 (72–390) in pAS2-1
–Trp
9.4 TRP1, ampr
pTD1
GAL4 2H
pTD1-1
GAL4 2H-2
pVA3
GAL4 2H
pVA3-1
GAL4 2H-2
a
LexA/GAL4 fusion gene,
–His
~13.5
HIS3, ampr pLexA-Pos
LexA 2H
Iwabuchi et al., 1993 Bartel et al., 1993a
Clontech
Hind III
(4.6, 2.2, 1.7, 0.9)
Hind III
(4.6, 1.8)
Hind III
(7.3, 1.2, 1.0, 0.5)
Hind III
(12, 1.3, 1.2, 0.5)
Hind III
(6.0, 4.5, 3.0)
Iwabuchi et al., 1993
Chien et al., 1991
Iwabuchi et al., 1993
Li & Fields, 1993;
Li & Fields, 1993;
Chien et al., 1991
Golemis et al., 1994
(pSH17-4)
Hind III
(5.2, 4.3, 0.9, 0.2)
Hind III
(5.7, 5.2, 0.2)
Hind III
(4.6, 2.2, 0.9, 0.85, 0.4)
Hind III
(4.7, 0.8, 0.6)
BamHI/EcoR I
(7.3, 0.57)
Clontech
Clontech
Fields & Song, 1989
Li & Fields, 1993;
Chien et al., 1991
Estojak et al., 1995
(pSH18-34)
References (Plasmid
name in reference)
pB42AD-T, pLAM5’, pLAM5’-1, pLexA-53, pLexA-Lam, pTD1-1, and pVA3-1 are derivatives of the plasmids described in the indicated references; plasmids were modified at
Clontech.
~10.0
Human lamin C(66–230) –His
10.6 in pLexA, HIS3, ampr
pLexA-Lam
LexA 2H
11.1 Human lamin C(66–230) –Trp
6.0 in pGBT9, TRP1, ampr
pLAM5’
GAL4 2H
murine p53(72–390) in pLexA
–His
HIS3, ampr
Human lamin C(66-230) –Trp
7.9
in pGBKT7, TRP1, kanr
pGBKT7-Lam
GAL4 2H-3
LexA 2H
murine p53(72-390) in pGBKT7,
–Trp
8.3
TRP1, kanr
pGBKT7-53
GAL4 2H-3
pLexA-53
Xho I/EcoR I
(8.0, 2.0)
10.0
in pGADT7, LEU2, ampr
pGADT7-T
GAL4 2H-3
BamHI/EcoR I
(7.3, 1.0)
Hind III
(~11.2, 2.8, 1.8)
Hind III
(3.4, 2.1, 1.0, 0.9, 0.6, 0.5)
pCL1
GAL4 2H & 2H-2 wild-type full-length GAL4
–Leu
~15.3 gene in a YCp50 derivative,
LEU2, ampr
SV40 large T-antigen(87–708) –Trp
8.5
in pB42AD, TRP1, ampr
pB42AD-T
LexA 2H
Hind III
(6.3, 2.1, 1.9)
Size
(kb)
Diagnostic R.E. Sites (kb)
lacZ under control of –Ura
~10.3 lexAop(x8), URA3, ampr Selection on SD Medium
Matchmaker two-hybrid system reporter and control plasmids
p8op-lacZ
LexA 2H
Vectora
System
Description
APPENDIX E: Plasmid Information continued
61
5.4 6.9 HIS3 under control of
–Hise
cloned target element, ampr lacZ under control of
–Ura
cloned target element, URA3, ampr
pHISi-1
pLacZi
EcoR I/Xho I
(6.4, 0.04)f
EcoR I/Xho I
(4.4, 1.0)
EcoR I/Xho I
(5.7, 1.0)f
Luo et al., 1996
Alexandre et al., 1993
Alexandre et al., 1993
Luo et al., 1996
Luo et al., 1996
Luo et al., 1996
a
In the one-hybrid system, a putative recognition sequence (the target DNA sequence) must be cloned into the MCS of one of the reporter plasmids. The construct is then used to
generate the necessary yeast reporter strain for detecting specific DNA-protein interactions.
b
Additional vector information, restriction maps, and multiple cloning site (MCS) sequences are provided in the Matchmaker One-Hybrid System User Manual.
c
p53HIS and p53Blue are not cut by Sma I, which makes it possible to distinguish them from pHISi and pLacZi, each of which have a single Sma I site.
d
pGAD53m is not cut by Xho I.
e
Leaky HIS3 expression in these plasmids permits its use as a selectable marker on SD/–His (without 3-AT).
f
In addition, pHISi and pLacZi have a single Sma I site, which makes it possible to distinguish them from p53HIS and p53Blue, which are not cut by Sma I.
6.8 HIS3 under control of
–Ura, –Hise
cloned target element, URA3, ampr pHISi
(7.6)
EcoR Id
7.6 murine p53(72–300) fused to
GAL4(768–881) AD, LEU2, ampr
pGAD53m
–Leu
EcoR I/Xho Ic
(6.3, 0.1)
lacZ under control of
–Ura
6.4 p53 binding sites in pLacZi, URA3, ampr
p53BLUE
EcoR I/Xho Ic
(5.7, 1.0)
HIS3 under control of
–Ura, –His
6.7 p53 binding sites in pHISi,
HIS3, URA3, ampr
p53HIS
Diagnostic
R.E. Sites
(kb)
Reference
Matchmaker one-hybrid system cloning, reporter & control plasmids
Selection on Size
Vectora
Descriptionb
SD Medium
(kb)
table viii.
APPENDIX E: Plasmid Information continued
www.clontech.com
table ix. yeast reporter strains in the
www.clontech.com
The trp1, his3, gal4, and gal80 mutations are all deletions; leu2–3, 112 is a double mutation. The LYS2 gene is nonfunctional in the HF7c and CG-1945. See Chapter II for more
information on the promoters of the reporter genes.
b
See Table V for more information on reporter genes and their phenotypes.
c
Genes that are used as selection markers in this system.
d
PT Libraries = Pretransformed Matchmaker Libraries.
e
CG-1945 is a derivative of HF7c (Feilotter et al., 1994).
f
The ade2–101 gene of the precursor strain was replaced (by recombination) with the GAL2-ADE2 reporter construct.
a
James et al., 1996;
Holtz, Unpublished
MATa, trp1-901, leu2-3, 112, ura3-52, his3-200,
gal4Δ, gal80Δ, LYS2 : : GAL1UAS-GAL1TATA-HIS3, MEL1 GAL2UAS-GAL2TATA-ADE2, URA3::MEL1UAS-MEL1TATA-lacZ
AH109f
GAL4 2H-3
HIS3, ADE2, lacZ,
trp1, leu2
MEL1
MATa, trp1-901, leu2-3, 112, ura3-52, his3-200,
HIS3, ADE2, MEL1
trp1, ura3, leu2
James et al., 1996
gal4Δ, gal80Δ, LYS2 : : GAL1UAS-GAL1TATA-HIS3, GAL2UAS-GAL2TATA-ADE2, MEL1
PJ69-2Af
PT Libraries
MATa, ura3-52, his3-200, lys2-801, ade2-101, ade5,
his3, trp1
Liu et al., 1993
trp1-901, leu2-3, 112, tyr1-501,gal4Δ, gal80Δ, ade5 : : hisG Estojak et al., 1995 LexA 2H & MM 1H
his3, trp1, ura3
YM4271
LEU2
MATα, ura3, his3, trp1, LexAop (x6)-LEU2
trp1, leu2,
Harper et al., 1993;
cyhr2
Flick & Johnston, 1990
LexA 2H
MATa, ura3-52, his3-200, ade2-101, lys2-801, HIS3, lacZ, MEL1
trp1-901, leu2-3, 112, gal4Δ, gal80Δ, cyhr2, LYS2 : : GAL1UAS-HIS3TATA-HIS3, MEL1 URA3 : : GAL1UAS-GAL1TATA-lacZ
Y190
GAL4 2H-2
trp1, leu2,
Feilotter et al., 1994;
cyhr2 C. Giroux, personal communication
EGY48
MATa, ura3-52, his3-200, ade2-101, lys2-801,
HIS3, lacZ
trp1-901, leu2-3, 112, gal4-542, gal80-538, cyhr2,
LYS2 : : GAL1UAS-GAL1TATA-HIS3, URA3 : : GAL4 17-mers(x3)-CYC1TATA-lacZ
GAL4 2H-2,
MATα, ura3-52, his3-200, ade 2-101,
lacZ, MEL1
trp1, leu2
Harper et al., 1993
GAL4 2H-3, & trp 1-901, leu 2-3, 112, gal4Δ, met–, gal80Δ, PT Librariesd URA3 : : GAL1UAS-GAL1TATA-lacZ, MEL1
CG-1945e
GAL4 2H-2,
Y187
MATa, ura3-52, his3-200, ade2-101, lys2-801, HIS3, lacZ
trp1-901, leu2-3, 112, gal4-542, gal80-538, LYS2 : : GAL1UAS-GAL1TATA-HIS3, URA3 : : GAL4 17-mers(x3)-CYC1TATA-lacZ
HF7c
GAL4 2H
Harper et al., 1993
References
trp1, leu2,
Feilotter et al., 1994;
cyhr2 C. Giroux, personal communication
MATa, ura3-52, his3-200, ade2-101, lys2-801, lacZ
trp1, leu2
trp 1-901, leu2-3, 112, canr, gal4-542, gal80-538, URA3 : : GAL1UAS-GAL1TATA-lacZ
SFY526
GAL4 2H
Transformation
Markersc
Matchmaker one-and two-hybrid systems
Reporter(s)b
Strain
System
Genotypea
APPENDIX F: Yeast Host Strain Information
63
Notes
AmpliTaq® and GeneAmp® are registered trademarks of Hoffmann-LaRoche, Inc., and F. Hoffmann LaRoche, Ltd.
FalconTM is a trademark of Becton Dickinson & Co.
Galacton-StarTM and Sapphire IITM are trademarks of Tropix, Inc.
GenBankTM is a trademark of the U.S. Department of Health & Human Services.
Parafilm® is a registered trademark of the American Can Co.
www.clontech.com

Yeast Protocols Handbook User Manual FOR RESEARCH USE ONLY PT3024-1 (PR973283)

Transcription

Similar documents

FLEISCHMANN`S YEAST IS ON THE RISE

Red Yeast Rice - Biotics Research Corporation

Recommended by Autism Specialists to Address

Frequent Sufferer

Yeast Infections - Health Momentum

Points to Remember

Vaginal Infection

Red Yeast Rice - Biotics Research Corporation